18f labeling of proteins using sortases

ABSTRACT

The present invention, in some aspects, provides methods, reagents, compositions, and kits for the radiolabeling of proteins, for example, of proteins useful for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (e.g., for diagnostic and therapeutic applications), using sortase-mediated transpeptidation reactions. Some aspects of this invention provide methods for the conjugation of an agent, for example, a radioactive agent or molecule to diagnostic or therapeutic peptides or proteins. Compositions comprising sortagged, radiolabeled proteins as well as reagents for generating radiolabeled proteins are also provided. Kits comprising reagents useful for the generation of radiolabeled proteins are provided, as are precursor proteins that comprise a sortase recognition motif.

RELATED APPLICATIONS

This application is a divisional of application of U.S. patentapplication U.S. Ser. No. 15/035,924, filed May 11, 2016, which isnational stage filing under 35 U.S.C. § 371 of international PCTapplication, PCT/US2014/065574, filed Nov. 13, 2014, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication, U.S. Ser. No. 61/903,834, filed Nov. 13, 2013, each ofwhich is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI087879, GM106409, and GM100518 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Positron emission tomography (PET) is a powerful technology for medicaland biological imaging and the scope of PET applications is expandingrapidly. The development of suitable PET tracers is critical to PETtechnology. An increasing number of PET tracers are peptide/proteinbased and are useful to specifically label tissues in therapeutic and/ordiagnostic applications.

Fluorine-18 (¹⁸F) is a short-lived isotope of fluorine with suitableproperties for PET-imaging. A majority of presently usedradiopharmaceuticals in PET are labeled with fluorine-18. However, sincethe half-life of ¹⁸F isotope is only about 110 minutes, there iscurrently no facile and general method to efficiently andsite-specifically modify proteins with ¹⁸F or other radionuclides in ashort time period.

SUMMARY OF THE INVENTION

Aspects of the present disclosure relate to the recognition thatefficient and facile means of labeling peptides and proteins fortherapeutic and diagnostic applications are needed. In the context ofPET or single-photon emission computed tomography (SPECT) diagnosticimaging utilizing peptide or protein-based tracers, quickly generatingradiolabeled peptides/proteins is prerequisite to the use of suchtracers given the short half-life of commonly used radioisotopes such as¹⁸F. Surprisingly, as disclosed herein, sortagging (sortase-mediatedtranspeptidation) of proteins of interest using novel radiolabeledsortase substrates allows for a robust and efficient way of generatingradiolabeled proteins, which are site-specifically labeled. Suchmethodology is useful, e.g., for quickly generatingpeptide/protein-based PET tracers. Other aspects of the disclosure arebased on the knowledge that there exist commercially availableradioactive agents used in PET or other imaging modalities, and usingsuch agents as a radiation source for labeling peptides or proteins ofinterest using sortagging technology can reduce the amount of timeinvolved in preparing radiolabeled peptide/protein tracers and/or reducethe amount of time between the preparation and administration ofpeptide/protein tracers. However, other sources of radiation (e.g.,non-commercially available compounds/reagents) are amenable to use withthe methods, compositions, reagents, systems, and kits provided herein.

Accordingly, certain aspects of the invention provide methods forlabeling a protein having a sortase recognition motif, with aradiolabel. Typically, the methods comprise contacting the protein witha radiolabeled sortase substrate peptide in the presence of a sortaseunder conditions suitable for the sortase to transamidate the proteinand the sortase substrate peptide. In some embodiments, the methodcomprises contacting the protein with a sortase substrate peptide in thepresence of a sortase under conditions suitable for the sortase totransamidate the protein and the sortase substrate peptide, therebyproducing a modified protein, wherein the sortase substrate peptidecomprises a click chemistry handle (e.g., tetrazine ortrans-cyclooctene); and contacting the modified protein with aradiolabeled agent, wherein the radiolabeled agent comprises acomplementary click chemistry handle (e.g., tetrazine ortrans-cyclooctene), which reacts with the click chemistry handle of themodified protein, thereby producing a radiolabeled protein. The proteincomprises either a C-terminal or N-terminal sortase recognition motif,and the sortase substrate peptide comprises a complementary sortaserecognition motif. For example, in some embodiments the proteincomprises a C-terminal sortase recognition motif (e.g., LPXTG, where Xis any amino acid), and the sortase substrate peptide comprises acomplementary N-terminal sortase recognition motif (e.g., GGG). In someembodiments, prior to being conjugated to the protein of interest, thesortase substrate peptide is linked to a radiolabeled agent through useof nucleophile/electrophile pairings, chelation (e.g., NOTA) or clickchemistry. In some embodiments, the sortase substrate peptide is linkedto the agent via an oxime linkage, a hydrazone linkage, athiosemicarbazone linkage, an amide linkage, an ester linkage, an etherlinkage, a disulfide linkage, a click chemistry linkage, or othersuitable linkage. In other embodiments, the sortase substrate peptide isfirst tethered to a protein of interest using a sortase-mediatedtranspeptidation reaction and subsequently modified to incorporate thelabel (e.g., a radiolabel, such as ¹⁸F). For example, a sortasesubstrate peptide comprising a click-chemistry handle may be conjugatedto a protein of interest using a sortase mediated transpeptidationreaction. The protein of interest, containing the click chemistryhandle, may subsequently be labeled (e.g., with a radiolabel) using anysuitable click chemistry reaction known in the art. In some embodiments,the C-terminal sortase recognition motif is LPXTX or NPXTX, wherein eachinstance of X independently represents any amino acid residue. Forexample, in some embodiments, the C-terminal sortase recognition motifis LPETG (SEQ ID NO:2), LPETA (SEQ ID NO:3), NPQTN (SEQ ID NO:4), orNPKTG (SEQ ID NO:5). In some embodiments, the N-terminal recognitionmotif comprises an oligoglycine or an oligoalanine sequence, for example1-10 N-terminal glycine residues (e.g., GGG) or 1-10 N-terminal alanineresidues (e.g., AAA), respectively. In some embodiments, the sortasesubstrate peptide comprises the sequence (G)_(n1)K, wherein n1 is aninteger between 1 and 10, inclusive. The radiolabeled sortase substratepeptide comprises one or more radionuclide(s) suitable for diagnosticand/or therapeutic applications, such as PET. For example, in someembodiments, the radionuclide is carbon-11, carbon-14, nitrogen-13,oxygen-15, fluorine-18, rubidium-82, copper-61, copper-62, copper-64,yttrium-86, gallium-68, zirconium-89, or iodine-124. In someembodiments, the radiolabeled agent linked to the sortase substratepeptide is a radiolabeled carbohydrate, for example, a sugar such asfludeoxyglucose (¹⁸F-FDG) or ¹⁴C—(U)-glucose. In some embodiments, theradiolabeled agent linked to the sortase substrate peptide is ¹⁸F, whichin some embodiments is sourced or derived from sodium fluoride(¹⁸F—NaF). ¹⁸F—NaF can be used as a source of ¹⁸F for labeling proteinsand sortase substrate peptides using a substitution reaction where onefunctional group in a chemical compound is replaced by anotherfunctional group. In certain embodiments, the sortase used totransamidate the protein and sortase substrate peptide is sortase A,sortase B, sortase C or sortase D. In some embodiments, the sortase usedto transamidate the protein and sortase substrate peptide is sortase Afrom Staphylococcus aureus (SrtA_(aureus)), sortase A from Streptococcuspyogenes (SrtA_(pyogenes)), sortase B from S. aureus (SrtB_(aureus)),sortase B from Bacillus anthracis (SrtB_(anthracis)), sortase B fromListeria monocytogenes (SrtB_(monocytogenes)), sortase C fromEnterococcus faecalis (SrtCfaecalis), sortase C from Streptococcusagalactiae (SrtC_(agalactiae)) sortase C from Streptococcuspneumonia(SrtCpneumonia), sortase C from Actinomyces oris (SrtC_(oris)),sortase C from Streptococcus suis (SrtC_(suis)), or sortase D fromBacillus cereus (SrtD_(cereus)). In some embodiments, the methods allowfor the fast and efficient conjugation of the protein to the sortasesubstrate peptide. For example, in some embodiments, the protein isconjugated to the sortase substrate peptide in less than 5 minutes, lessthan 10 minutes, less than 15 minutes, less than 20 minutes, less than25 minutes, less than 30 minutes, less than 45 minutes, less than 60minutes, less than 90 minutes, or less than 120 minutes. Further, insome embodiments, at least 50%, at least 60%, at least 75%, at least90%, at least 95%, or at least 98% of the protein is labeled with thesortase substrate peptide. In some embodiments, the protein to beconjugated with a radiolabeled sortase substrate peptide is a proteinsuitable for diagnostic and/or therapeutic applications (e.g., PET orSPECT imaging), such as an antibody, an antibody fragment, an affibody,a single-domain antibody, a Fab fragment, or a therapeutic peptide. Insome embodiments, the protein binds to a tumor cell, a tumor-associatedcell, or a tumor antigen. In other embodiments, the protein binds to animmune cell, such as a T-cell, a B-cell, a plasma cell, a macrophage, adendritic cell, a neutrophil, an eosinophil, or a mast cell. In someembodiments, the protein binds to a marker of inflammation (e.g., MHCclass II molecules, CD3, CD4, CD8, CD11b). In some embodiments, themethod further involves purifying the labeled (e.g., conjugated)protein, resulting, in some embodiments, in a composition of thepurified protein that comprises at least 10, at least 20, at lease 30,at least 40, at least 50, at least 75, at least 100, at least 150, atleast 200, at least 250, at least 300, at least 350, at least 400, atleast 450, at least 500, at least 550, at least 600, at least 700, atleast 800, at least 900, or at least 1000 MBq of radioactivity.

According to another aspect, compositions suitable for use in themethods, described herein, (e.g., methods for radiolabeling a proteinusing a sortase) are provided. In some embodiments, the compositions foruse in the methods, described herein, include a radiolabeled sortasesubstrate peptide, a sortase, and a protein comprising a sortaserecognition motif. In other embodiments, the compositions for use in themethods, described herein, include a protein that has beensite-specifically conjugated to a click chemistry handle, and aradiolabel conjugated to a click chemistry handle. In yet otherembodiments the compositions for use in the methods, described herein,include a sortase substrate peptide comprising a click chemistry handle,a sortase, a protein comprising a sortase recognition motif and aradiolabel conjugated to a click chemistry handle. It should beappreciated that the compositions useful for the methods, describedherein, (e.g., for site-specifically radiolabeling a protein) may beprovided in a kit. In some embodiments, the protein comprises aC-terminal sortase recognition motif (e.g., as described herein), andthe sortase substrate peptide comprises an N-terminal recognition motif(e.g., as described herein). The sortase substrate peptide comprises aradionuclide, for example, as part of an agent linked to the peptide,which is suitable for diagnostic and/or therapeutic applications asdescribed herein. In some embodiments, the composition further comprisesa sortase as described herein. The protein in the composition is anyprotein suitable for the diagnostic and/or therapeutic applicationsdescribed herein (e.g., an antibody, an antibody fragment, an affibody,a single-domain antibody, a Fab fragment, or a therapeutic peptide), forexample, those that bind to a tumor cell, a tumor-associated cell, or atumor antigen.

According to another aspect, compositions comprising radiolabeledproteins are provided. In some embodiments, the radiolabeled protein isgenerated according to the methods (e.g., methods for radiolabelingproteins using sortases) provided herein. It should be appreciated thatthe site-specific conjugation of a protein to a peptide or other moiety(e.g., a radiolabeled agent) may be achieved using other enzymes knownin the art. For example, formylglycine generating enzyme,phosphopantetheinyltransferases, transglutaminase, farnesyltransferase,biotin ligase, lipoic acid ligase, or N-myristoyl transferase. In someembodiments, the protein is radiolabeled with a radionuclide (e.g.,carbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82,copper-61, copper-62, copper-64, yttrium-86, gallium-68, zirconium-89,iodine-124, etc.) suitable for diagnostic and/or therapeuticapplications described herein. In some embodiments, the protein islabeled (using a sortase substrate peptide) with a sugar, such asfludeoxyglucose (¹⁸F-FDG) or ¹⁴C—(U)-glucose. In other embodiments, ¹⁸Fsodium fluoride (¹⁸F—NaF) is used to radiolabel an agent via asubstitution reaction that comprises a click chemistry handle having asuitable leaving group. In some embodiments, the radiolabeled clickchemistry handle can be used to label a protein having a complementaryclick chemistry handle that has been site-specifically conjugated to theprotein. In some embodiments, the protein is any protein suitable forthe diagnostic and/or therapeutic applications described herein (e.g.,an antibody, antibody fragment, an affibody, a single-domain antibody, aFab fragment, or a therapeutic peptide), for example, those that bind toa tumor cell, a tumor-associated cell, a tumor antigen, or a marker ofinflammation. In some embodiments, the composition comprises at least10, at least 20, at lease 30, at least 40, at least 50, at least 75, atleast 100, at least 150, at least 200, at least 250, at least 300, atleast 350, at least 400, at least 450, at least 500, at least 550, atleast 600, at least 700, at least 800, at least 900, or at least 1000MBq of radioactivity. In some embodiments, the composition is suitablefor administration to a subject (e.g., a subject undergoing diagnosticand/or therapeutic procedure(s) described herein), and further comprisesa pharmaceutically acceptable carrier. In some embodiments, thecompositions suitable for administration to a subject (e.g.,site-specifically radiolabeled proteins), described herein, aresubstantially pure. In some embodiments the compositions are at least75%, at least 80%, at least 85%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or at least 99.5% pure.In yet other embodiments the compositions, suitable for administrationto a subject, as described herein, are at least 90% pure.

According to yet another aspect, methods for modifying a sortasesubstrate peptide are provided. Such methods are useful for generatingradiolabeled sortase substrate peptides which are suitable for use inthe methods described herein (e.g., methods for labeling a protein witha radiolabeled sortase substrate peptide). Typically, the methodcomprises contacting a sortase substrate peptide that comprises anucleophilic group (e.g., an aminooxy group, a hydrazide,thiosemicarbazide, or click chemistry handle, e.g., trans-cyclooctene)with a radiolabeled agent that comprises an electrophilic group (e.g., acarbonyl-containing functional group or click chemistry handle, e.g.,tetrazine) under conditions suitable for the formation of a covalentbond between the sortase substrate peptide and agent. In someembodiments, the agent comprises a radionuclide (e.g., carbon-11,carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82, copper-61,copper-62, copper-64, yttrium-86, gallium-68, zirconium-89, iodine-124,etc.) suitable for a diagnostic and/or therapeutic application describedherein. In some embodiments, the agent is a sugar, such asfludeoxyglucose (¹⁸F-FDG) or ¹⁴C—(U)-glucose. In other embodiments, theagent comprises ¹⁸F (e.g., sourced from ¹⁸F sodium fluoride (¹⁸F—NaF).In some embodiments, the sortase substrate peptide comprises anN-terminal sortase recognition motif, e.g., an oligoglycine or anoligoalanine sequence, for example 1-10 N-terminal glycine residues(e.g., GGG) or 1-10 N-terminal alanine residues (e.g., AAA),respectively. In some embodiments, the sortase substrate peptidecomprises the sequence (G)_(n1)K, wherein n1 is an integer between 1 and10, inclusive. In some embodiments, the K is modified to include anucleophilic group (e.g., an aminooxy group). In some embodiments, themethods allow for the fast and efficient modification of a sortasesubstrate peptide, thus in some embodiments, the method furthercomprises contacting the sortase substrate peptide and/or agent with acatalyst. In some embodiments, the catalyst is m-phenylenediamine(mPDA), o-phenylenediamine, p-phenylenediamine, o-aminophenol,m-aminophenol, p-aminophenol, o-aminobenzoic acid, 5-methoxyanthranilicacid, 3,5-diaminobenzoic acid, or aniline. In some embodiments, thesortase substrate peptide is modified in less than 5 minutes, less than10 minutes, less than 15 minutes, less than 20 minutes, less than 25minutes, less than 30 minutes, less than 45 minutes, less than 60minutes, less than 90 minutes, or less than 120 minutes. Further, insome embodiments, at least 50%, at least 60%, at least 75%, at least90%, at least 95%, or at least 98% of the sortase substrate peptide iscovalently linked to the agent.

According to another aspect, sortase substrate peptides linked to aradiolabeled agent are provided. In some embodiments, the sortasesubstrate peptide is generated according to methods provided herein. Insome embodiments, the sortase substrate peptide and agent are linked byan oxime, a hydrazone, a thiosemicarbazone, or a click chemistrylinkage. In some embodiments, the radiolabeled agent comprises aradionuclide (e.g., carbon-11, carbon-14, nitrogen-13, oxygen-15,fluorine-18, rubidium-82, copper-61, copper-62, copper-64, yttrium-86,gallium-68, zirconium-89, iodine-124) suitable for the diagnostic and/ortherapeutic applications described herein. In some embodiments, theagent is a sugar, such as fludeoxyglucose (¹⁸F-FDG) or ¹⁴C—(U)-glucose.In some embodiments, the sortase substrate peptide comprises anN-terminal sortase recognition motif, e.g., an oligoglycine or anoligoalanine sequence, for example, 1-10 N-terminal glycine residues(e.g., GGG) or 1-10 N-terminal alanine residues (e.g., AAA),respectively. In some embodiments, the sortase substrate peptidecomprises the sequence (G)_(n1)K, wherein n1 is an integer between 1 and10, inclusive.

In yet another aspect, compositions comprising a sortase substratepeptide, a radiolabeled agent, and a catalyst are provided. Suchcompositions are useful for generating radiolabeled sortase substratepeptides according to the methods provided herein. In some embodiments,the sortase substrate peptide comprises an N-terminal sortaserecognition motif, e.g., an oligoglycine or an oligoalanine sequence,for example, 1-10 N-terminal glycine residues (e.g., GGG) or 1-10N-terminal alanine residues (e.g., AAA), respectively. In someembodiments, the sortase substrate peptide comprises the sequence(G)_(n1)K, wherein n1 is an integer between 1 and 10, inclusive. In someembodiments, the K is modified to include a nucleophilic group (e.g., anaminooxy group). In some embodiments, the radiolabeled agent comprises aradionuclide (e.g., carbon-11, carbon-14, nitrogen-13, oxygen-15,fluorine-18, rubidium-82, copper-61, copper-62, copper-64, yttrium-86,gallium-68, zirconium-89, iodine-124, etc.) suitable for the diagnosticand/or therapeutic applications described herein. In some embodiments,the agent is a sugar, such as fludeoxyglucose (¹⁸F-FDG) or¹⁴C—(U)-glucose. In some embodiments, the catalyst is m-phenylenediamine(mPDA), o-phenylenediamine, p-phenylenediamine, o-aminophenol,m-aminophenol, p-aminophenol, o-aminobenzoic acid, 5-methoxyanthranilicacid, 3,5-diaminobenzoic acid, or aniline.

According to another aspect, methods of diagnosing, monitoring, and/ortreating a subject using the inventive compositions are provided.Typically, the method comprises: (a) administering an inventivecomposition (e.g., a radiolabeled protein generated according to themethods provided herein) to the subject; and (b) detecting theradiolabel in the subject. In some embodiments, the subject has, hashad, or is suspected of having cancer. In some embodiments, the subjecthas, has had, or is suspected of having a proliferative disease. In someembodiments, the subject has, has had, or is suspected of having aninflammatory disease or disorder. In some embodiments, detecting theradiolabel is performed using positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT).

In another aspect, kits are provided. For example, kits useful forcarrying out any of the inventive methods or for generating any of theinventive compositions are provided. In some embodiments, a kit formodifying a sortase substrate peptide comprising a sortase substratepeptide (e.g., a sortase substrate peptide comprising a nucleophilicgroup such as a click chemistry handle, an aminooxy group, a hydrazide,or thiosemicarbazide) a modifying agent (e.g., a radioactive agent thatcomprises an electrophilic group such as an click chemistry handle or acarbonyl-containing group (e.g., ¹⁸F-FDG or ¹⁴C—(U)-glucose)) and acatalyst (e.g., m-phenylenediamine (mPDA), o-phenylenediamine,p-phenylenediamine, o-aminophenol, m-aminophenol, p-aminophenol,o-aminobenzoic acid, aniline, etc.), are provided. In some embodiments,a kit (e.g., for labeling a protein) is provided that comprises aradiolabeled sortase substrate peptide, e.g., generated according to theinventive methods described herein. In some embodiments, the kitcomprises a sortase, such as sortase A, sortase B, sortase C, or sortaseD. Some non-limiting examples of sortases that may be used are sortase Afrom Staphylococcus aureus (SrtA_(aureus)), sortase A from Streptococcuspyogenes (SrtA_(pyogenes)), sortase B from Staphylococcus aureus(SrtB_(aureus)), sortase B from Bacillus anthracis (SrtB_(anthracis)),sortase B from Listeria monocytogenes (SrtB_(monocytogenes)), sortase Cfrom Enterococcus faecalis (SrtC_(faecalis)), sortase C fromStreptococcus agalactiae (SrtC_(agalactiae)) sortase C fromStreptococcus pneumonia (SrtC_(pneumonia)), sortase C from Actinomycesoris (SrtC_(oris)), sortase C from Streptococcus suis (SrtC_(suis)), orsortase D from Bacillus cereus (SrtD_(cereus)). In some embodiments, akit is provided that comprises a radiolabeled protein (e.g., anantibody, an antibody fragment, an affibody, a single-domain antibody, aFab fragment, or a therapeutic peptide) generated according to theinventive methods described herein.

The above summary is intended to provide an overview of some aspects ofthis invention and is not to be construed to limit the invention in anyway. Additional aspects, advantages, and embodiments of this inventionare described herein, and further embodiments will be apparent to thoseof skill in the art based on the instant disclosure. The entire contentsof all references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the dynamic equilibrium between cyclic hemiacetal andlinear aldehyde forms of glucose (FIG. 1A) the modification of a sortasesubstrate peptide (FIG. 1B), and identification of sortagged protein(VHH4) using the modified substrate (FIGS. 1C-1D). (FIG. 1B) Schematicrepresentation of the strategy to produce glucose-labeled protein usingsortagging technology. First the sortase substrate peptide (1) islabeled via oxime ligation with glucose using m-Phenylenediamine (mPDA)as the catalyst. Next, the product (2) is added to the sortaggingreaction mixture which contains sortase and VHH4. The enzyme andnon-sortagged protein is separated from the sortagged protein byincubating the solution with Ni-NTA beads. The product is analyzed byLC-MS analysis. (FIG. 1C) LC-MS analysis of the starting protein,VHH4-LPETGGHis6. (FIG. 1D) LC-MS analysis of the glucose-labeledprotein, VHH4-LPET-peptide 2.

FIGS. 2A-2C show an exemplary site-specific labeling of VHH4 proteinwith fludeoxyglucose (FDG) using sortagging technology. First thesortase substrate peptide 1 is labeled via oxime ligation with FDG usingmPDA as the catalyst. Next, the product (2) is added to the sortaggingsolution. The enzyme and non-sortagged protein is separated from thesortagged protein by incubating the solution with Ni-NTA beads. Theproduct is analyzed with LC-MS analysis. (FIG. 2B) LC-MS analysis of thestarting protein, VHH4-LPETGGHis6. (FIG. 2C) LC-MS analysis of thelabeled protein, VHH4-LPET-peptide-FDG.

FIGS. 3A-3C show the site-specific labeling of VHH4 with ¹⁴C—(U)-glucoseusing sortagging. (FIGS. 3A and 3B) Schematic representation of thereactions. (FIG. 3C) SDS-PAGE analysis of the labeling reaction. Theband correlating with ¹⁴C-radiolabeled VHH4 is indicated.

FIGS. 4A-4H show a schematic representation of the strategy forsite-specific labeling of proteins with ¹⁸F using sortagging and thetetrazine (Tz)/trans-cyclooctene (TCO) reverse-electron demandDiels-Alder cycloaddition reaction. (FIG. 4A) A protein equipped at itsC-terminus with the LPXTG sortase-recognition motif, followed by a tag(e.g., 6×His) is incubated with sortase A, which cleaves thethreonine-glycine bond and via its active site cysteine residue forms areactive acyl intermediate. Addition of a peptide comprising a series ofN-terminal glycine residues (e.g., three (3) glycines) and a functionalmoiety of choice results in site-specific modification of the protein.(FIG. 4B) A protein equipped with a sortase recognition motif ismodified with a (Gly)₃-R, e.g., where R is a tetrazine (Tz) derivative.(FIG. 4C) The formation of site-specifically modified protein isconfirmed by LC-MS: shown here for VHH7-Tz, a single domain antibodyspecific for murine Class II MHC molecules, as an example. (FIG. 4D)Tosyl-trans-cyclooctene (TCO) and ¹⁸F—NaF are used to produce ¹⁸F-TCOand purified by HPLC. ¹⁸F-TCO is added to the Tz-modified VHH, and thereaction is allowed to proceed for ˜20 min. The ¹⁸F-labeled VHH isquickly separated from free label by desalting on a PD-10 columnpre-equilibrated with PBS, yielding a radiolabeled protein solutionready for injection. (FIG. 4E) The sortase reaction was used to installa high affinity copper chelating, 1,4,7-triazacyclononane-triacetic acid(NOTA), molecule at the C-terminus of a VHH protein followed by additionof ⁶⁴Cu²⁺ to produce ⁶⁴Cu-VHH. (FIG. 4F) The NOTA-labeled VHH wasconfirmed by LC-MS. (FIG. 4G) Radio-TLC analysis of 18F-VHHs. Radio-TLCanalysis of labeled VHHs was performed after size-exclusionchromatography demonstrating >98% radiochemical purity of all labeledVHHs. (FIG. 4H) Radio-TLC analysis of 64Cu-VHHs. Radio-TLC analysis oflabeled VHHs was performed after size-exclusion chromatographydemonstrating >98% radiochemical purity of all labeled VHHs.

FIGS. 5A-5B show that ¹⁸F-VHH7 (anti mouse Class II MHC) detectssecondary lymphoid organs. (FIG. 5A) PET images of C57BL/6 (right) andClass II MHC^(−/−) (left) mice 2 hours post-injection of ¹⁸F-VHH7. (FIG.5B) PET-CT 3D-rendering of C57BL/6 mouse 2 hours post-injection of¹⁸F-VHH7. Accumulation of ¹⁸F-VHH7 is shown in lymph nodes (bilaterallysymmetrical; numbers 1, 2, 4 and 5) and thymus (3), superimposed onribcage, along the body axis.

FIGS. 6A-6D show the pharmacokinetic profile of ¹⁸F-VHH7. (FIG. 6A)PET-derived standardized uptake values (SUVs) for different tissues fora C57BL/6 mouse 2.5 hours post-injection. (FIG. 6B) Biodistribution of¹⁸F-VHH7 in C57BL/6 and MHC-II^(−/−) mice 2.5 hours post-injection.(FIG. 6C) Blood half-life measurement in the C57BL/6 mouse. Percent ofinjected dose per gram of blood (% ID/g) was measured at different timesusing decay-corrected intensities of collected bloods. (FIG. 6D) Datawas fit to a two-compartment model (bi-exponential non-linearregression) to give a weighted blood half-life of 6.0 min.

FIGS. 7A-7J depict imaging the presence of tumor-associated Class IIMHC⁺ cells using ¹⁸F-VHH7 (anti mouse Class II MHC). A NOD-SCID mousewas inoculated subcutaneously with human MelJuSo melanoma cells andimaged 25 days post injection. Tumor cells lack mouse class II MHCmolecules. (FIGS. 7A, 7B and 7C) Coronal PET-CT images, moving anteriorto posterior. In (FIG. 7A) and (FIG. 7B), different sets of bilaterallysymmetrically disposed lymph nodes are visible. In (FIG. 7C),tumor-associated Class II MHC positive cells are visible, attributableto influx of host-derived Class II MHC positive cells. (FIG. 7D) PET-CTas maximum intensity projections of all slices. (FIG. 7E & FIG. 7F)¹⁸F-VHH7 detects Class II MHC⁺ cells associated with small tumors atearly stages. NOD-SCID mice were inoculated with Mel-Juso cells as in(FIG. 7A), 20 (FIG. 7E) and 6 (FIG. 7F) days prior to imaging.Transverse PET and CT images (left and right, respectively) are shownfor better visualization of the Class II MHC⁺ cells at the site ofcancer cells. Images are all window-leveled to the same intensity.Tumors and associated Class II MHC⁺ cells are highlighted with arrows.Axillary (A), brachial (B) and mediastinal (M) lymph nodes and thymus(T) are shown in (FIG. 7F). (G&H) A NOD-SCID mouse was inoculatedsubcutaneously on the back of the neck with 5×10⁶ human Mel-Jusomelanoma cells and imaged 30 days post injection with ¹⁸F-VHHDC13.Tumor-associated CD11b positive cells are visible, attributable toinflux of host-derived CD11b positive cells. (FIG. 7I & FIG. 7J) FACSanalysis of tumor-infiltrating immune cells. The next day following theimaging, tumors were excised and digested with collagenase D, andtumor-infiltrating cells were obtained after Percoll gradient. Cellsuspensions were then stained for FACS analysis. (FIG. 71) Histogramsshow the FACS staining with the VHH7 of mouse CD45⁺ tumor-infiltratingcells. Histograms on the left are gated on CD11c⁺CD11b⁺ cells (dendriticcells) and histograms on the right are gated on CD11c⁻CD11b⁺ cells(macrophages and other myeloid cells) for the indicated time points.(FIG. 7J) Tumor-infiltrating cells were harvested 30 days after tumorinoculation as in (FIG. 71) and stained with VHHDC13. Histograms showthe levels of CD11b as measured by VHHDC13 on the indicated cellpopulations. Spleen from the same mouse is shown for comparison. Forleft panel (spleen): gray, red and black represent CD11b⁻, CD11b⁺CD11c⁺and CD11b⁺CD11c⁻, respectively. For the right panel (tumor): the left isCD45−CD11b− and the peak on the right is CD45⁺CD11b⁺. Experiments arerepresentative of two mice for each time point and FACS staining.

FIGS. 8A-8F show that ⁶⁴Cu-VHH7 (anti mouse Class II MHC) and⁶⁴Cu-VHHDC13 (myeloid cell-specific) detect secondary lymphoid organsand inflammation. (FIGS. 8A, 8B and 8C) PET images of C57BL/6 mouse 4 h,8 h, and 24 h post-injection of ¹⁸F-VHH7, respectively, demonstratingspecificity for Class II MHC organs. FIG. 8E) PET image of C57BL/6 mouse4 h post-injection of ¹⁸F-VHHDC13, demonstrating specificity for myeloidcells. (FIGS. 8D and 8F) Complete Freund's adjuvant (CFA) was injectedto the left paw of C57BL/6 mice and ⁶⁴Cu-VHH7 (for FIG. 8D) or⁶⁴Cu-VHHDC13 (for FIG. 8F) was used to image inflammation 24 h after CFAinjection. Images were obtained 4 h post injection of ⁶⁴Cu-VHHs;inflammation around the injection site is clearly visible, attributableto influx of host-derived Class II⁺ or myeloid cells for (FIG. 8D) and(FIG. 8F), respectively (arrows). Images are all window-leveled to thesame intensity for better comparison.

FIGS. 9A-9B show 18F-VHH7 (anti mouse Class II MHC) and 18F-VHH DC13(anti CD11b) detect secondary lymphoid organs and inflammation. (FIGS.9A and 9B) Complete Freund's adjuvant (CFA) was injected to the left pawof C57BL/6 mice and 18F-VHHDC13 (for FIG. 9A) or 18F-VHH7 (for FIG. 9B)was used to image their targets 24 h after CFA injection. PET-CT Imageswere obtained 1.5 h post injection of 18F-VHHs; Images are allwindow-leveled to the same intensity for comparison.

FIGS. 10A-10C show a schematic representation of the strategy forsite-specific labeling of proteins with ¹⁸F using a pre-preparedsortagged protein and ¹⁸F-FDG oxime-tetrazine. (FIG. 10A) ¹⁸FDG is addedto a solution of tetrazine-aminooxy and a catalyst, p-phenylenediamine,to produce ¹⁸F-FDG oxime-tetrazine. (FIG. 10B) Excess tetrazine-aminooxycan be captured by adding the highly water soluble sugar, glucosamine6-sulfate. The ¹⁸F-FDG oxime-tetrazine can then be purified from therest of the reaction mixture due to the change in hydrophilicity. (FIG.10C) First, (1) A single domain antibody fragment (VHH) equipped at itsC-terminus with the LPXTG sortase recognition motif is linked to a(glycine)₃-TCO using a sortase to generate a VHH having the TCO clickchemistry handle. Second, (2) the ¹⁸F-FDG oxime-tetrazine generated in(FIG. 10B) is added to the VHH that has been sortagged to TCO to createan ¹⁸F-labeled VHH.

FIG. 11 shows a scheme for the synthesis of of N-succinimidyl4-[18F]fluorobenzoate (SFB). The reaction conditions are as follows: (A)Kryptofix 222, [18F]fluoride, DMSO, 120-140° C.; B) KMnO4, NaOH, 120°C.; C) DSC, pyridine, CH3CN, 150° C. (Vaidyanathan et al., NatureProtocols, 2006, (1), 1655-1661).

FIGS. 12A-12E show a schematic of site-specific 18F or 64Cu-labeling ofsingle domain antibodies (VHHs) using sortase. (FIG. 12A) A VHH having asortase recognition sequence (LPXTG) is contacted with a sortasesubstrate peptide having three glycines (G)₃ conjugated to a tetrazinederivative in the presence of sortase to produce a VHH having asite-specific tetrazine click-chemistry handle. (FIG. 12B)Tosyl-trans-cyclooctene (TCO) and ¹⁸F—NaF are used to produce ¹⁸F-TCOand purified by HPLC. (FIG. 12C) The VHH having a site-specifictetrazine click-chemistry handle is reacted to the ¹⁸F-TCO to generate aVHH that is site-specifically labeled with 18F. (FIG. 12D) The formationof site-specifically 18Fmodified VHH is confirmed by LC-MS. (FIG. 12E)The sortase reaction was used to install a high affinity copperchelating, 1,4,7-triazacyclononane-triacetic acid (NOTA), molecule atthe C-terminus of a VHH protein followed by addition of ⁶⁴Cu²⁺ toproduce VHH-⁶⁴Cu. The ⁶⁴Cu in this figure may be replaced with ⁶⁸Ga.

FIG. 13 shows tetrazine-amine (left) reacted with 18F-SFB to producetetrazine-18F. The product may be reacted with a site specificTCO-labeled protein to form the final 18F-labeled protein.

FIG. 14 shows exemplary application of the radioactive protein andcompositions thereof.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

In a formula, - - - is absent, a coordination bond between a ligand anda metal, or a single bond.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, theterms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms (C₁₋₂₀ aliphatic). In certain embodiments, the aliphaticgroup has 1-10 carbon atoms (C₁₋₁₀ aliphatic). In certain embodiments,the aliphatic group has 1-6 carbon atoms (C₁₋₆ aliphatic). In certainembodiments, the aliphatic group has 1-5 carbon atoms (C₁₋₅ aliphatic).In certain embodiments, the aliphatic group has 1-4 carbon atoms (C₁₋₄aliphatic). In certain embodiments, the aliphatic group has 1-3 carbonatoms (C₁₋₃ aliphatic). In certain embodiments, the aliphatic group has1-2 carbon atoms (C₁₋₂ aliphatic). Aliphatic group substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms (C₁₋₂₀ alkyl). In anotherembodiment, the alkyl group employed contains 1-15 carbon atoms(C₁₋₁₅alkyl). In another embodiment, the alkyl group employed contains1-10 carbon atoms (C₁₋₁₀alkyl). In another embodiment, the alkyl groupemployed contains 1-8 carbon atoms (C₁₋₈ alkyl). In another embodiment,the alkyl group employed contains 1-6 carbon atoms (C₁₋₆ alkyl). Inanother embodiment, the alkyl group employed contains 1-5 carbon atoms(C₁₋₅alkyl). In another embodiment, the alkyl group employed contains1-4 carbon atoms (C₁₋₄alkyl). In another embodiment, the alkyl groupemployed contains 1-3 carbon atoms (C₁₋₃ alkyl). In another embodiment,the alkyl group employed contains 1-2 carbon atoms (C₁₋₂ alkyl).Examples of alkyl radicals include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substituents. Alkyl group substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety. The term “alkylene,” as used herein,refers to a biradical derived from an alkyl group, as defined herein, byremoval of two hydrogen atoms. Alkylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Alkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀ alkenyl). In some embodiments, thealkenyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅ alkenyl). In another embodiment, the alkenyl group employedcontains 2-10 carbon atoms (C₂₋₁₀ alkenyl). In still other embodiments,the alkenyl group contains 2-8 carbon atoms (C₂₋₈ alkenyl). In yet otherembodiments, the alkenyl group contains 2-6 carbons (C₂₋₆ alkenyl). Inyet other embodiments, the alkenyl group contains 2-5 carbons (C₂₋₅alkenyl). In yet other embodiments, the alkenyl group contains 2-4carbons (C₂₋₄ alkenyl). In yet other embodiments, the alkenyl groupcontains 2-3 carbons (C₂₋₃ alkenyl). In yet other embodiments, thealkenyl group contains 2 carbons (C₂alkenyl). Alkenyl groups include,for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike, which may bear one or more substituents. Alkenyl groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety. Theterm “alkenylene,” as used herein, refers to a biradical derived from analkenyl group, as defined herein, by removal of two hydrogen atoms.Alkenylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkenylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀alkynyl). In some embodiments, thealkynyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅ alkynyl). In another embodiment, the alkynyl group employedcontains 2-10 carbon atoms (C₂₋₁₀ alkynyl). In still other embodiments,the alkynyl group contains 2-8 carbon atoms (C₂₋₈ alkynyl). In stillother embodiments, the alkynyl group contains 2-6 carbon atoms (C₂₋₆alkynyl). In still other embodiments, the alkynyl group contains 2-5carbon atoms (C₂₋₅ alkynyl). In still other embodiments, the alkynylgroup contains 2-4 carbon atoms (C₂₋₄ alkynyl). In still otherembodiments, the alkynyl group contains 2-3 carbon atoms (C₂₋₃ alkynyl).In still other embodiments, the alkynyl group contains 2 carbon atoms(C₂alkynyl). Representative alkynyl groups include, but are not limitedto, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like, which maybear one or more substituents. Alkynyl group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety. The term “alkynylene,” asused herein, refers to a biradical derived from an alkynylene group, asdefined herein, by removal of two hydrogen atoms. Alkynylene groups maybe cyclic or acyclic, branched or unbranched, substituted orunsubstituted. Alkynylene group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms andfor 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”). Insome embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”). Insome embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms include, withoutlimitation, triazinanyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetra-hydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14 π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14 π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

The term “unsaturated” or “partially unsaturated” refers to a moietythat includes at least one double or triple bond.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups are optionally substituted. “Optionally substituted”refers to a group which may be substituted or unsubstituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, and includes any of the substituents described herein thatresults in the formation of a stable compound. The present inventioncontemplates any and all such combinations in order to arrive at astable compound. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety. The invention is notintended to be limited in any manner by the exemplary substituentsdescribed herein.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino. In certain embodiments, the “substitutedamino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb) C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined herein. Nitrogen protecting groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc),vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate(Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-dcyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-cliphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

The terms “aminooxy,” or “aminooxy group,” are used interchangeablyherein and refer to functional groups having the general formula:

wherein R³¹ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³¹ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “tautomers” or “tautomeric” refers to two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a single bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim,enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

The terms “carbonyl,” or “carbonyl group,” are used interchangeablyherein and refer to functional groups composed of a carbon atomdouble-bonded to any oxygen atom. Carbonyls have the general formula:

wherein each of R³² and R³³ independently represents hydroxyl,optionally substituted amino, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl. Examples of carbonyls include, but are not limited to,aldehydes, ketones, carboxylic acids, esters, amides, enones, acylhalides, acid anhydrides, and imides. In some embodiments, acarbonyl-containing compound refers to a compound having an aldehydegroup, or a compound capable of forming an aldehyde group throughisomerization. For example, in some embodiments, certain sugars (e.g.,reducing sugars) such as glucose, form aldehydes through isomerization.A sugar is classified as a reducing sugar if it has an open-chain formwith an aldehyde group or a free hemiacetal group. Monosaccharides whichcontain an aldehyde group are known as aldoses, and those with a ketonegroup are known as ketoses. The aldehyde can be oxidized via a redoxreaction in which another compound is reduced. Thus, a reducing sugar isone that is capable of reducing certain chemicals. Sugars with ketonegroups in their open chain form are capable of isomerizing via a seriesof tautomeric shifts to produce an aldehyde group in solution.Therefore, ketone-bearing sugars like fructose are considered reducingsugars but it is the isomer containing an aldehyde group which isreducing since ketones cannot be oxidized without decomposition of thesugar. This type of isomerization is catalyzed by the base present insolutions which test for the presence of aldehydes.

The term “hydrazide,” as used herein, refers to functional groups havingthe general formula:

wherein R³⁴ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³⁴ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “hydrazone,” as used herein, refers to compound having thegeneral formula:

wherein each of R³⁵, R³⁶, and R³⁷ is independently optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. The term “hydrazone linkage,” asused herein, refers to the formula:

Hydrzones can be prepared from, for example, joining of a compoundcomprising a hydrazide group and a compound comprising a carbonyl.

The term “acyl,” as used herein, is a subset of a substituted alkylgroup, and refers to a group having the general formula —C(═O)R^(A),—C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A), or —C(═O)N(R^(A))₂,wherein each instance of R^(A) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “azide” or “azido,” as used herein, refers to a group of theformula (—N₃).

The term “agent,” as used herein, refers to any molecule, entity, ormoiety that can be conjugated to a sortase recognition motif, a sortasesubstrate peptide, or any other enzymatic recognition motif orenzcymatic substrate peptide known in the art. For example, an agent maybe a protein, an amino acid, a peptide, a polynucleotide, acarbohydrate, a detectable label, a tag, a metal atom, a contrast agent,a non-polypeptide polymer, a synthetic polymer, a recognition element, alipid, or chemical compound, such as a small molecule. In someembodiments, the agent is radioactive or comprises a radiolabel. In someembodiments, the agent is enriched for a particular isotope of anelement. In some embodiments, the agent comprises a radionuclide (e.g.,a radioactive atom) or isotope selected from the group consisting ofcarbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82,copper-61, copper-62, copper-64, yttrium-86, gallium-68, zirconium-89,and iodine-124. The agent may connected to a radionuclide by a carbonbond, for example, an ¹⁸F may be linked to the agent by a C-¹⁸F bond. Insome embodiments, the agent is a carbonyl- (e.g., an aldehyde- orketone-) containing group that comprises a radionuclide. In someembodiments, the agent is fludeoxyglucose (¹⁸F-FDG). In otherembodiments, the agent is an alkene (e.g., a TCO) that comprises aradionuclide, such as ¹⁸F. Radiolabeling of tosyl-trans-coclooctene(TCO) with ¹⁸F can be achieved with [¹⁸F]—F/K222/K₂CO₃ in DMSO forapproximately 10 minutes at 90° C. to produce 18F-TCO (Keliher, E. J. etal. “Two-Step 18F Labeling Strategy for 18F-PARP1 Inhibitors.”,ChemMedChem 6, 424-427, 2011) and purified by HPLC. In some embodiments,the agent is ¹⁴C—(U)-glucose. In some embodiments, the agent cannot begenetically encoded. In some such embodiments, the agent is a lipid, anamino acid, a nucleotide, a carbohydrate, or a small molecule. Forexample, in some embodiments, the agent is a radioactive carbohydrate,which in addition to FDG and ¹⁴C—(U)-glucose, includes, but is notlimited to, 2-deoxy-2-fluoro-D-mannose [¹⁸F];6-deoxy-6-fluoro-D-fructose [¹⁸F]; citric acid [1,5-¹⁴C];deoxy-D-glucose, 2-[1-¹⁴C], galactose 1-phosphate D-[¹⁴C(U)]; galactose,D-[1-¹⁴C]; glucosamine hydrochloride D-[6-³H(N)]; glucosamine,N-acetyl-D-[1-¹⁴C]; glucose 1-phosphate, α-D-[¹⁴C(U)]; glucose,3-0-[methyl-D-1-³H]; glucose, D-[6-¹⁴C]; glucose, L-[1-¹⁴C]; glycerol,[¹⁴C(U)]; inositol, Myo-[2-³H(N)]; inulin-carboxyl, [carboxyl-¹⁴C];inulin-methoxy, [methoxy-³H]; lactose, [D-glucose-1-¹⁴C]; mannitol,D-[1-¹⁴C]; mannose, D-[2-³H(N)]; methyl α-D-glucopyrano side,[glucose-¹⁴C(U)]; methyl-D-glucose, 3-0-[methyl-¹⁴C]; myo-inositol,[³H]; starch, [¹⁴C(U)]; uridine diphospho-D-glucose, [6-³H]; and xylose,D-[U-¹⁴C]. The particular isotope used to label any of the foregoingradioactive carbohydrates can be substituted with any other isotopedescribed herein. In some embodiments, the radioactive agent is a smallmolecule or compound, which include, but is not limited to, acetic acid,−[1-¹⁴C]; benzylamine HCL, [7-¹⁴C]; biotin, [8,9-³H(N)]-(VitaminH);choline chloride, [methyl-¹⁴C]; D-(+) Biotin, [³H(G)]; ethyl maleimide,N-[ethyl-1-¹⁴C]; ketoglutaric acid, α-[1-¹⁴C]; lactic acid, L-[¹⁴C(U)];malic acid, L-[1,4(2,3)-¹⁴C]; mephenytoin, S[4-¹⁴C];methyl-tetrahydrofolic acid, 5-[¹⁴C]-barium salt, NAD, [carbonyl-¹⁴C];nociceptin, [leucyl-3,4,5-³H]; NSP [³H]—,(N-succininidyl[2,3-³H]propionate); polyethylene glycol, [1,2-³H];pyruvic acid, [1-¹⁴C]; sodium bicarbonate [¹⁴C]; tetraethylammoniumbromide, [1-¹⁴C]; urea, [14C]; adenosine 3″,5″-cyclic phosphoric acid,2″-O-succinyl, [¹²⁵I]-iodotyrosine methyl ester; and deoxyuridine[¹²⁵I]-iodo-2″. The particular isotope used to label any of theforegoing radioactive small molecules or compounds can be substitutedwith any other isotope described herein. In some embodiments, theradioactive agent is a lipid, which includes, but is not limited to,3-indolylacetic acid, [5-³H(N)]; 5-hydroxy tryptamine, [³H]; acetylcoenzyme A [acetyl-1-¹⁴C]; arachidonic acid [1-¹⁴C]; carnitinehydrochloride, L-[N-methyl-¹⁴C]; cholesteryl hexadecyl ether,[cholesteryl-1,2-³H(N)]; choline chloride, [methyl-³H]; farnesylpyrophosphate, triammonium salt, [1-³H(N)]; geranylgeranylpyrophosphate, [1-3^(H)(N)]glycerol [2-³H]; glycerol 3-phosphate,ammonium salt, L-[¹⁴C(U)]; glycerol tri oleate, [1-¹⁴C]; glycerol trioleate, [9,10(N)-³H]; hydroxy-3-methylglutaryl coenzyme A,DL-3-[glutaryl-3-¹⁴C]; hydroxycholesterol, 25-[26,27-³H]; iloprost,[³H]; inositol-1,4,5-triphosphate, D-[inositol-1-³H(N)]; isopentenylpyrophosphate, [4-¹⁴C]; leukotriene [14,15,19,20-³H(N)]; linoleic acid,[1-¹⁴C]; lysopalmitoyl phosphatidylcholine, L-1-[palmitoyl-1-¹⁴C];lysophosphatidic acid, 1-Oleoyl-[oleoyl-9,10-³H]; mevalonolactone,RS-[2-¹⁴C]; myristic acid (tetradecanoic acid), [9,10-³H(N)]; oleic acid[1-¹⁴C]; oleoyl coenzyme A [oleoyl-1-¹⁴C]; palmitic acid [1-¹⁴C];palmitoyl carnitine chloride, L-[palmitoyl-1-¹⁴C]; palmitoyl coenzyme A,[palmitoyl-1-¹⁴C]; phenylethylamine hydrochloride, beta-ethyl 1,¹⁴C;phosphatidic acid, L-α-dipalmitoyl-[glycerol-¹⁴C(U)];phosphatidylcholine [dipalmitoyl-1-¹⁴C]; phosphatidylinositol[MYO-inositol-2-³H(N)]; hexadecylPAF,1-O-hexadecyl-[acetyl-³H(N)]-[acetyl-³H]; retinoic acid,[11,12-³H(N)]; retinol, [11,12-³H(N)]; sphingomyelin[choline-methyl-¹⁴C]; stearic acid, [1-¹⁴C]; taurine, [2,2,³H];taurocholic acid, [carbonyl-¹⁴C]; triolein [9 10-³H(N)]; and verapamilhydrochloride, [N-Methyl-³H]. The particular isotope used to label anyof the foregoing radioactive lipids can be substituted with any otherisotope described herein. In some embodiments, the agent is aradioactive nucleotide, which includes, but is not limited to, adenine,[2,8-³H]; adenosine 5′-diphosphate [8-¹⁴C]; adenosine 5′-triphosphate,[2,5′,8-³H]; caffeine, [1-methyl-¹⁴C]; cytidine 5′-triphosphate, [5-³H];deoxy guanosine 5′-triphosphate, [8-³H(N)]—, (dGTP, [8-³H(N)];deoxycytidine 5-triphosphate 5,5-³H; deoxythymidine 5′-triphosphate[methyl-³H]; deoxyuridine 5′-triphosphate, [5-³H(N)]-(dUTP [5-³H(N)]);guano sine 5′-triphosphate, [8-3H], (GTP,[³H]); hypoxanthinemonohydrochloride, ³H; nitrobenzylthioinosine, [benzyl-³H]; thymidine[2-¹⁴C]; thymidine, [6-3H]; uracil, 5,6,³H; uridine diphosphate glucose,[glucose-¹⁴C(U)]; and uridine, [U-14C]. The particular isotope used tolabel any of the foregoing radioactive nucleotides can be substitutedwith any other isotope described herein. Additional agents suitable foruse in embodiments of the present invention will be apparent to theskilled artisan. The invention is not limited in this respect.

The term “amino acid,” as used herein, includes any naturally occurringand non-naturally occurring amino acid. There are many known non-naturalamino acids any of which may be included in the polypeptides or proteinsdescribed herein. See, for example, S. Hunt, The Non-Protein AminoAcids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C.Barrett, Chapman and Hall, 1985. Some non-limiting examples ofnon-natural amino acids are 4-hydroxyproline, desmosine,gamma-aminobutyric acid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and para-substituted phenylalanines (e.g., substituted with —C(═O)C₆H₅;—CF₃; —CN; -halo; NO₂; —CH₃), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with —C(═O)C₆H₅; —CF₃; —CN; -halo;—NO₂; —CH₃), and statine. In the context of amino acid sequences, “X” or“Xaa” represents any amino acid residue, e.g., any naturally occurringand/or any non-naturally occurring amino acid residue.

The term “antibody”, as used herein, refers to a protein belonging tothe immunoglobulin superfamily. The terms antibody and immunoglobulinare used interchangeably. With some exceptions, mammalian antibodies aretypically made of basic structural units each with two large heavychains and two small light chains. There are several different types ofantibody heavy chains, and several different kinds of antibodies, whichare grouped into different isotypes based on which heavy chain theypossess. Five different antibody isotypes are known in mammals, IgG,IgA, IgE, IgD, and IgM, which perform different roles, and help directthe appropriate immune response for each different type of foreignobject they encounter. In some embodiments, an antibody is an IgGantibody, e.g., an antibody of the IgG1, 2, 3, or 4 human subclass.Antibodies from mammalian species (e.g., human, mouse, rat, goat, pig,horse, cattle, camel) are within the scope of the term, as areantibodies from non-mammalian species (e.g., from birds, reptiles,amphibia) are also within the scope of the term, e.g., IgY antibodies.

Only part of an antibody is involved in the binding of the antigen, andantigen-binding antibody fragments, their preparation and use, are wellknown to those of skill in the art. As is well-known in the art, only asmall portion of an antibody molecule, the paratope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). Suitable antibodies andantibody fragments for use in the context of some embodiments of thepresent invention include, for example, human antibodies, humanizedantibodies, domain antibodies, F(ab′), F(ab′)₂, Fab, Fv, Fc, and Fdfragments, antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; antibodies in which the FR and/or CDR1 and/orCDR2 and/or light chain CDR3 regions have been replaced by homologoushuman or non-human sequences; antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; and antibodies in which the FRand/or CDR1 and/or CDR2 regions have been replaced by homologous humanor non-human sequences. In some embodiments, so-called single chainantibodies (e.g., ScFv), (single) domain antibodies, and otherintracellular antibodies may be used in the context of the presentinvention. Domain antibodies, camelid and camelized antibodies andfragments thereof, for example, VHH domains, or nanobodies, such asthose described in patents and published patent applications of AblynxNV and Domantis are also encompassed in the term antibody. Further,chimeric antibodies, e.g., antibodies comprising two antigen-bindingdomains that bind to different antigens, are also suitable for use inthe context of some embodiments of the present invention. In someembodiments, the term antibody may also refer to “antibody mimetics,”which are organic compounds the can specifically bind antigens, but arenot structurally related to antibodies. For example, antibody mimeticsknown as “affibodies,” or “affibody molecules,” are small proteinsengineered to bind e.g., target proteins or peptides with affinitiescomparable to monoclonal antibodies. In some embodiments, an affibodyincludes a protein scaffold based on the Z domain (the immunoglobulin Gbinding domain) of protein A, and in contrast to antibodies, affibodymolecules are composed of alpha helices and lack disulfide bridges.Methods for engineering and producing affibodies are known, and includethose described in Nord et al., “A combinatorial library of an a-helicalbacterial receptor domain.” Prot. Eng. 1995; 8 (6): 601-608; Nord etal., “Binding proteins selected from combinatorial libraries of anα-helical bacterial receptor domain.” Nature Biotechnol. 1997; 15 (8):772-777; Ståhl et al., “The use of gene fusions to protein A and proteinG in immunology and biotechnology.” Pathol. Biol. (Paris) 1997; 45 (1):66-76; Rönnmark et al., “Construction and characterization ofaffibody-Fc chimeras produced in Escherichia coli.” J. Immunol. Methods.2002; 261 (1-2): 199-211; Rönnmark et al., “Affibody-beta-galactosidaseimmunoconjugates produced as soluble fusion proteins in the Escherichiacoli cytosol.” J. Immunol. Methods. 2003; 281 (1-2): 149-160; Nord etal., “Recombinant human factor VIII-specific affinity ligands selectedfrom phage-displayed combinatorial libraries of protein A.” Eur. J.Biochem. 2001; 268 (15): 1-10; Engfeldt et al., “Chemical synthesis oftriple-labeled three-helix bundle binding proteins for specificfluorescent detection of unlabeled protein.” Chem. BioChem. 2005; 6 (6):1043-1050; Ahlgren et al., “Targeting of HER2-expressing tumors with asite-specifically 99mTc-labeled recombinant affibody molecule,ZHER2:2395, with C-terminally engineered cysteine.” J. Nucl. Med. 2009;50 (5): 781-789; Orlova et al., “Evaluation of[(111/114m)In]CHX-A”-DTPA-ZHER2:342, an affibody ligand conjugate fortargeting of HER2-expressing malignant tumors.” Q. J. Nucl. Med. Mol.Imaging. 2007; 51 (4): 314-23; Tran et al., “(99m)Tc-maEEE-Z(HER2:342),an Affibody molecule-based tracer for the detection of HER2 expressionin malignant tumors”. Bioconjug. Chem. 2007; 18 (6): 1956-64; Orlova etal., “Tumor imaging using a picomolar affinity HER2 binding affibodymolecule.” Cancer Res. 2006; 66 (8): 4339-48; Holm et al.,“Electrophilic Affibodies Forming Covalent Bonds to Protein Targets.”The Journal of Biological Chemistry 2009; 284 (47): 32906-13; Renberg etal., “Affibody molecules in protein capture microarrays: evaluation ofmultidomain ligands and different detection formats.” J. Proteome Res.2007; 6 (1): 171-179; Lundberg et al., “Site-specifically conjugatedanti-HER2 Affibody molecules as one-step reagents for target expressionanalyses on cells and xenograft samples.” J. Immunol. Methods 2007; 319(1-2): 53-63; Tolmachev et al., “Radionuclide therapy of HER2-positivemicroxenografts using a 177Lu-labeled HER2-specific Affibody molecule.”Cancer Res. 2007; 67 (6): 2773-82; and Gebauer & Skerra, “Engineeredprotein scaffolds as next-generation antibody therapeutics.” CurrentOpinion in Chemical Biology 2009; 13 (3): 245-55; Siontorou C.,“Nanobodies as novel agents for disease diagnosis and therapy.” Int. J.Nanomedicine 2013; 8: 4215-4227; the entire contents of each are herebyincorporated by reference in their entirety.

The term “antigen-binding antibody fragment,” as used herein, refers toa fragment of an antibody that comprises the paratope, or a fragment ofthe antibody that binds to the antigen the antibody binds to, withsimilar specificity and affinity as the intact antibody. Antibodies,e.g., fully human monoclonal antibodies, may be identified using phagedisplay (or other display methods such as yeast display, ribosomedisplay, bacterial display). Display libraries, e.g., phage displaylibraries, are available (and/or can be generated by one of ordinaryskill in the art) that can be screened to identify an antibody thatbinds to an antigen of interest, e.g., using panning. See, e.g., Sidhu,S. (ed.) Phage Display in Biotechnology and Drug Discovery (DrugDiscovery Series; CRC Press; 1^(st) ed., 2005; Aitken, R. (ed.) AntibodyPhage Display: Methods and Protocols (Methods in Molecular Biology)Humana Press; 2nd ed., 2009.

The term “binding agent,” as used herein, refers to any molecule thatbinds another molecule with high affinity. In some embodiments, abinding agent binds its binding partner with high specificity. Examplesfor binding agents include, without limitation, antibodies, antibodyfragments, nucleic acid molecules, receptors, ligands, aptamers, andadnectins.

The term “click chemistry” refers to a chemical philosophy introduced byK. Barry Sharpless of The Scripps Research Institute, describingchemistry tailored to generate covalent bonds quickly and reliably byjoining small units comprising reactive groups together (see H. C. Kolb,M. G. Finn and K. B. Sharpless (2001). Click Chemistry: Diverse ChemicalFunction from a Few Good Reactions. Angewandte Chemie InternationalEdition 40 (11): 2004-2021. Click chemistry does not refer to a specificreaction, but to a concept including, but not limited to, reactions thatmimic reactions found in nature. In some embodiments, click chemistryreactions are modular, wide in scope, give high chemical yields,generate inoffensive byproducts, are stereospecific, exhibit a largethermodynamic driving force to favor a reaction with a single reactionproduct, and/or can be carried out under physiological conditions. Insome embodiments, a click chemistry reaction exhibits high atom economy,can be carried out under simple reaction conditions, use readilyavailable starting materials and reagents, uses no toxic solvents oruses a solvent that is benign or easily removed (preferably water),and/or provides simple product isolation by non-chromatographic methods(crystallisation or distillation). In some embodiments, the clickchemistry reaction is a [2+3] dipolar cycloaddition. In certainembodiments, the click chemistry reaction is a Diels-Aldercycloaddition.

The term “click chemistry handle,” as used herein, refers to a reactant,or a reactive group, that can partake in a click chemistry reaction.Exemplary click chemistry handles are demonstrated in U.S. PatentPublication 20130266512, which is incorporated by reference herein. Forexample, a strained alkyne, e.g., a cyclooctyne, is a click chemistryhandle, since it can partake in a strain-promoted cycloaddition (see,e.g., Table 1). In general, click chemistry reactions require at leasttwo molecules comprising click chemistry handles that can react witheach other. Such click chemistry handle pairs that are reactive witheach other are sometimes referred to herein as partner click chemistryhandles. For example, an azide is a partner click chemistry handle to acyclooctyne or any other alkyne. Exemplary click chemistry handlessuitable for use according to some aspects of this invention aredescribed herein, for example, in Tables 1 and 2. In some embodiments,the click chemistry partners are a conjugated diene and an optionallysubstituted alkene, In other embodiments, the click chemistry partnersare an optionally substituted tetrazine and an optionally substitutedtrans-cyclooctene (TCO). In some embodiments, the click chemistrypartners are optionally substituted tetrazine (Tz) and optionallysubstituted trans-cyclooctene (TCO). Tz and TCO react with each other ina reverse-electron demand Diels-Alder cycloaddition reaction (See e.g.,Example 2, FIG. 4; Blackman et al., “The Tetrazine Ligation: FastBioconjugation based on Inverse-electron-demand Diels-Alder Reactivity.”J. Am. Chem. Soc. 2008; 130, 13518-13519). In other embodiments, theclick chemistry partners are an optionally substituted alkyne and anoptionally substituted azide. For example, a difluorinated cyclooctyne,a dibenzocyclooctyne, a biarylazacyclooctynone, or a cyclopropyl-fusedbicyclononyne can be paired with an azide as a click chemistry pair. Inother embodiments, the click chemistry partners are reactive dienes andsuitable tetrazine dienophiles. For example, TCO, norbornene, orbiscyclononene can be paired with a suitable tetrazine dienophile as aclick chemistry pair. In yet other embodiments, tetrazoles can act aslatent sources of nitrile imines, which can pair with unactivatedalkenes in the presence of ultraviolet light to create a click chemistrypair, termed a “photo-click” chemistry pair. The click chemistry pairmay also be a cysteine and a maleimide. For example the cysteine from apeptide (e.g., GGGC) may be reacted with a maleimide that is associatedwith a chelating agent (e.g., NOTA). Other suitable click chemistryhandles are known to those of skill in the art (See, e.g., Table 1;Spicer et al., “Selective chemical protein modification.” NatureCommunications. 2014; 5:4740). For two molecules to be conjugated viaclick chemistry, the click chemistry handles of the molecules have to bereactive with each other, for example, in that the reactive moiety ofone of the click chemistry handles can react with the reactive moiety ofthe second click chemistry handle to form a covalent bond. Such reactivepairs of click chemistry handles are well known to those of skill in theart and include, but are not limited to, those described in Table 1.

TABLE 1 Exemplary click chemistry handles and reactions. Exemplary rateconstant (M⁻¹s⁻¹)

1 × 10^(−3a) 1,3-dipolar cycloaddition

strain-promoted cycloaddition

Diels-Alder reaction

Thiol-ene reaction

8 × 10^(−2a) Strain-promoted cycloaddition

     2.3^(a) Strain-promoted cycloaddition

     1^(a) Strain-promoted cycloaddition

     0.1^(a) Strain-promoted cycloaddition

     9^(a) Inverse-electron demand Diels- Alder (IEDDA)

  17,500^(a)   35,000^(b) Inverse-electron demand Diels- Alder (IEDDA)

>50,000^(a)    880^(b) Inverse-electron demand Diels- Alder (IEDDA)

     0.9^(a) 1,3-dipolar cycloaddition (“photo-click”)

     58^(a) 1,3-dipolar cycloaddition (“photo-click”) Each of R⁴¹, R⁴²,and R⁴³ is indpendently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl. In certain embodiments, at least one of R⁴¹, R⁴², and R⁴³independently comprises a sortase recognition motif. In certainembodiments, one of R⁴¹, R⁴², and R⁴³ independently comprises a sortaserecognition motif. In certain embodiments, two of R⁴¹, R⁴², and R⁴³independently comprise a sortase recognition motif. In certainembodiments, each of R⁴¹, R⁴², and R⁴³ independently comprises a sortaserecognition motif. In some embodiments, at least one of R⁴¹, R⁴², andR⁴³ is independently R_(R)—LPXT—[X]_(y)—, wherein each occurrence of Xindependently represents any amino acid residue; each occurrence of y isan integer between 0 and 10, inclusive; and each occurrence of R_(R)independently represents a protein or an agent (e.g., a protein,peptide, a detectable label, a binding agent, a small molecule), and,optionally, a linker. Each instance of R₃ is independently H,substituted or unsubstituted alkyl (e.g., —CH₃), or substituted orunsubstituted aryl. ^(a)Exemplary rate constant for small-moleculemodels. ^(b)Exemplary on-protein rate constant.

In some embodiments, click chemistry handles used can react to formcovalent bonds in the absence of a metal catalyst. Such click chemistryhandles are well known to those of skill in the art and include theclick chemistry handles described in Becer, Hoogenboom, and Schubert(Table 2), “Click Chemistry beyond Metal-Catalyzed Cycloaddition,”Angewandte Chemie International Edition (2009) 48: 4900-4908:

TABLE 2 Exemplary click chemistry reactions. Reagent A Reagent BMechanism Notes on reaction^([a]) 0 azide alkyne Cu-catalyzed [3 + 2]azide- 2 h at 60° C. in H₂O alkyne cycloaddition (CuAAC) 1 azidecyclooctyne strain-promoted [3 + 2] azide- 1 h at RT alkynecycloaddition (SPAAC) 2 azide activated [3 + 2] Huisgen cycloaddition 4h at 50° C. alkyne 3 azide electron-deficient [3 + 2] cycloaddittion 12h at RT in H₂O alkyne 4 azide aryne [3 + 2] cycloaddition 4 h at RT inTHF with crown ether or 24 h at RT in CH₃CN 5 tetrazine alkeneDiels-Alder retro-[4 + 2] 40 min at 25° C. (100% yield) cycloaddition N₂is the only by-product 6 tetrazole alkene 1,3-dipolar cycloaddition fewmin UV irradiation and then (photoclick) overnight at 4° C. 7 dithiosterdiene hetero-Diels-Alder cycloaddition 10 min at RT 8 anthracenemaleimide [4 + 2] Diels-Alder reaction 2 days at reflux in toluene 9thiol alkene radical addition 30 min UV (quantitative conv.) or (thioclick) 24 h UV irradiation (>96%) 10 thiol enone Michael addition 24 hat RT in CH₃CN 11 thiol maleimide Michael addition 1 h at 40° C. in THFor 16 h at RT in dioxane 12 thiol para-fluoro nucleophilic substitutionovernight at RT in DMF or 60 min at 40° C. in DMF 13 amine para-fluoronucleophilic substitution 20 min MW at 95° C. in NMP as solvent ^([a])RT= room temperature, DMF = N,N-dimethylformamide, NMP =N-methylpyrolidone, THF = tetrahydrofuran, CH₃CN = acetonitrile.

Methods and compositions for using click chemistry in combination withsortagging technologies are known, and include those described by Ploeghet al., international PCT application, PCT/US2012/044584, filed Jun. 28,2012, published as WO 2013/003555 on Jan. 3, 2013; and Ploegh et al.,U.S. patent application U.S. Ser. No. 13/918,278, filed Jun. 14, 2013;the entire contents of each of which are incorporated herein byreference.

The term “conjugated” or “conjugation” refers to an association of twomolecules, for example, two proteins or a protein and an agent, e.g., asmall molecule, with one another in a way that they are linked by adirect or indirect covalent or non-covalent interaction. In certainembodiments, the association is covalent, and the entities are said tobe “conjugated” to one another. In some embodiments, a protein ispost-translationally conjugated to another molecule, for example, asecond protein, a small molecule, a detectable label, a click chemistryhandle, or a binding agent, by forming a covalent bond between theprotein and the other molecule after the protein has been formed, and,in some embodiments, after the protein has been isolated. In someembodiments, two molecules are conjugated via a linker connecting bothmolecules. For example, in some embodiments where two proteins areconjugated to each other to form a protein fusion, the two proteins maybe conjugated via a polypeptide linker, e.g., an amino acid sequenceconnecting the C-terminus of one protein to the N-terminus of the otherprotein. In some embodiments, two proteins are conjugated at theirrespective C-termini, generating a C—C conjugated chimeric protein. Insome embodiments, two proteins are conjugated at their respectiveN-termini, generating an N—N conjugated chimeric protein. In someembodiments, conjugation of a protein to a peptide is achieved bytranspeptidation using a sortase. See, e.g., Ploegh et al.,International PCT Patent Application, PCT/US2010/000274, filed Feb. 1,2010, published as WO/2010/087994 on Aug. 5, 2010, and Ploegh et al.,International Patent Application, PCT/US2011/033303, filed Apr. 20,2011, published as WO/2011/133704 on Oct. 27, 2011, the entire contentsof each of which are incorporated herein by reference, for exemplarysortases, proteins, recognition motifs, reagents, and methods forsortase-mediated transpeptidation. In other embodiments, conjugation ofa protein to a peptide or other moiety may be achieved using otherenzymes known in the art, for example, formylglycine generating enzyme,sialyltransferase, phosphopantetheinyltransferase, transglutaminase,farnesyltransferase, biotin ligase, lipoic acid ligase, or N-myristoyltransferase. Exemplary techniques and approaches for enzymatic labelingof proteins can be found in Rashidian, M., et al. “Enzymatic Labeling ofProteins: Techniques and Approaches”, Bioconjugate Chem., 2013; 24,1277-1294; which is incorporated by reference.

The term “detectable label” refers to a moiety that has at least oneelement or isotope incorporated into the moiety which enables detectionof the molecule, e.g., a protein or peptide, or other entity, to whichthe label is attached. Labels can be directly attached (i.e., via abond) or can be attached by a linker. It will be appreciated that thelabel may be attached to or incorporated into a molecule, for example, aprotein, polypeptide, a carbohydrate, or other entity, at any position.In some embodiments, a detectable label contains isotopic moieties,which may be radioactive or heavy isotopes, including, but not limitedto, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ⁶¹Cu, ⁶²Cu, ¹³N, ¹⁵N, ¹⁵O, ¹⁸F, ³¹P, ³²P,³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁵³Gd, ⁸⁹Sr, ⁸⁶Y, ¹⁶⁹Yb, ⁸²Rb, and ¹⁸⁶Re. In certain embodiments, alabel comprises a radioactive isotope, preferably an isotope which emitsdetectable particles, such as α particles, β particles or rays, such asγ rays.

The term “linker,” as used herein, refers to a chemical group ormolecule covalently linked to a molecule, for example, a protein, and achemical group or moiety, for example, a click chemistry handle. In someembodiments, the linker is positioned between, or flanked by, twogroups, molecules, or moieties and connected to each one via a covalentbond, thus connecting the two. In some embodiments, the linker is anamino acid or a plurality of amino acids. In some embodiments, thelinker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more than 20 amino acids. In some embodiments, thelinker comprises a poly-glycine sequence. In some embodiments, thelinker comprises a non-protein structure. In some embodiments, thelinker is an organic molecule, group, polymer, or chemical moiety (e.g.,poylethylene, polyethylene glycol).

The terms “nucleic acid” and “nucleic acid molecule,” as used herein,refer to a compound comprising a nucleobase and an acidic moiety, e.g.,a nucleoside, a nucleotide, or a polymer of nucleotides. Typically,polymeric nucleic acids, e.g., nucleic acid molecules comprising threeor more nucleotides are linear molecules, in which adjacent nucleotidesare linked to each other via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g. nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to an oligonucleotide chain comprising three or moreindividual nucleotide residues. As used herein, the terms“oligonucleotide” and “polynucleotide” can be used interchangeably torefer to a polymer of nucleotides (e.g., a string of at least threenucleotides). In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA. Nucleic acids may benaturally occurring, for example, in the context of a genome, atranscript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid,chromosome, chromatid, or other naturally occurring nucleic acidmolecule. On the other hand, a nucleic acid molecule may be anon-naturally occurring molecule, e.g., a recombinant DNA or RNA, anartificial chromosome, an engineered genome, or fragment thereof, or asynthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurringnucleotides or nucleosides. Furthermore, the terms “nucleic acid,”“DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e.analogs having other than a phosphodiester backbone. Nucleic acids canbe purified from natural sources, produced using recombinant expressionsystems, chemically synthesized, and, optionally, purified. Whereappropriate, e.g., in the case of chemically synthesized molecules,nucleic acids can comprise nucleoside analogs such as analogs havingchemically modified bases or sugars, and backbone modifications. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

The term “oxime,” as used herein, refers to compound having the generalformula:

wherein each of R⁴⁴, R⁴⁵, and R⁴⁶ is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. The term “oxime linkage,” as usedherein, refers to a linker comprising the O—N═C moiety. In certainembodiments, the oxime linkage can be formed by joining of a compoundcomprising an aminooxy group and a compound comprising a carbonyl.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as an aminooxy group, a hydrazidegroup, a thiosemicarbazide group, a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof.

The term “small molecule” is used herein to refer to molecules, whethernaturally-occurring or artificially created (e.g., via chemicalsynthesis) that have a relatively low molecular weight. Typically, asmall molecule is an organic compound (i.e., it contains carbon). Asmall molecule may contain multiple carbon-carbon bonds, stereocenters,and other functional groups (e.g., amines, hydroxyl, carbonyls,heterocyclic rings, etc.). In some embodiments, small molecules aremonomeric and have a molecular weight of less than about 1500 g/mol. Incertain embodiments, the molecular weight of the small molecule is lessthan about 1000 g/mol or less than about 500 g/mol. In certainembodiments, the small molecule is a drug, for example, a drug that hasalready been deemed safe and effective for use in humans or animals bythe appropriate governmental agency or regulatory body.

The term “sortase,” as used herein, refers to an enzyme able to carryout a transpeptidation reaction conjugating the C-terminus of a proteinor peptide to the N-terminus of a protein or peptide via transamidation.Sortases are also referred to as transamidases, and typically exhibitboth a protease and a transpeptidation activity. Various sortases fromprokaryotic organisms have been identified. For example, some sortasesfrom Gram-positive bacteria cleave and translocate proteins toproteoglycan moieties in intact cell walls. Among the sortases that havebeen isolated from Staphylococcus aureus, are sortase A (Srt A) andsortase B (Srt B). Thus, in certain embodiments, a transamidase used inaccordance with the present invention is sortase A, e.g., from S.aureus, also referred to herein as SrtA_(aureus). In certainembodiments, a transamidase is a sortase B, e.g., from S. aureus, alsoreferred to herein as SrtB_(aureus).

Sortases have been classified into four classes, designated A, B, C, andD, designated sortase A, sortase B, sortase C, and sortase D,respectively, based on sequence alignment and phylogenetic analysis of61 sortases from Gram-positive bacterial genomes (Dramsi S, Trieu-CuotP, Bierne H, Sorting sortases: a nomenclature proposal for the varioussortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005;the entire contents of which are incorporated herein by reference).These classes correspond to the following subfamilies, into whichsortases have also been classified by Comfort and Clubb (Comfort D,Clubb RT. “A comparative genome analysis identifies distinct sortingpathways in gram-positive bacteria” Infect Immun., 72(5):2710-22, 2004;the entire contents of which are incorporated herein by reference):Class A (Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3),Class D (Subfamilies 4 and 5). The aforementioned references disclosenumerous sortases and recognition motifs. See also Pallen, M. J.; Lam,A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3),97-101; the entire contents of which are incorporated herein byreference. Those skilled in the art will readily be able to assign asortase to the correct class based on its sequence and/or othercharacteristics such as those described in Drami, et al., supra. Theterm “sortase A” is used herein to refer to a class A sortase, usuallynamed SrtA in any particular bacterial species, e.g., SrtA from S.aureus. Likewise “sortase B” is used herein to refer to a class Bsortase, usually named SrtB in any particular bacterial species, e.g.,SrtB from S. aureus. The invention encompasses embodiments relating to asortase A from any bacterial species or strain. The inventionencompasses embodiments relating to a sortase B from any bacterialspecies or strain. The invention encompasses embodiments relating to aclass C sortase from any bacterial species or strain. The inventionencompasses embodiments relating to a class D sortase from any bacterialspecies or strain.

Amino acid sequences of Srt A and Srt B and the nucleotide sequencesthat encode them are known to those of skill in the art and aredisclosed in a number of references cited herein, the entire contents ofall of which are incorporated herein by reference. The amino acidsequence of a sortase-transamidase from Staphylococcus aureus also hassubstantial homology with sequences of enzymes from other Gram-positivebacteria, and such transamidases can be utilized in the ligationprocesses described herein. For example, for SrtA there is about a 31%sequence identity (and about 44% sequence similarity) with bestalignment over the entire sequenced region of the S. pyogenes openreading frame. There is about a 28% sequence identity with bestalignment over the entire sequenced region of the A. naeslundii openreading frame. It will be appreciated that different bacterial strainsmay exhibit differences in sequence of a particular polypeptide, and thesequences herein are exemplary.

In certain embodiments a transamidase bearing 18% or more sequenceidentity, 20% or more sequence identity, 30% or more sequence identity,40% or more sequence identity, or 50% or more sequence identity with anS. pyogenes, A. naeslundii, S. nutans, E. faecalis or B. subtilis openreading frame encoding a sortase can be screened, and enzymes havingtransamidase activity comparable to Srt A or Srt B from S. aureas can beutilized (e.g., comparable activity sometimes is 10% of Srt A or Srt Bactivity or more).

Thus in some embodiments of the invention the sortase is a sortase A(SrtA). SrtA recognizes the motif LPXTX (wherein each occurrence of Xrepresents independently any amino acid residue), with commonrecognition motifs being, e.g., LPKTG (SEQ ID NO:6), LPATG (SEQ IDNO:7), LPNTG (SEQ ID NO:8). In some embodiments LPETG (SEQ ID NO:2) isused as the sortase recognition motif. However, motifs falling outsidethis consensus may also be recognized. For example, in some embodimentsthe motif comprises an ‘A’ rather than a ‘T’ at position 4, e.g., LPXAG(SEQ ID NO:9), e.g., LPNAG (SEQ ID NO:10). In some embodiments the motifcomprises an ‘A’ rather than a ‘G’ at position 5, e.g., LPXTA (SEQ IDNO:11), e.g., LPNTA (SEQ ID NO:12), e.g., LPETA (SEQ ID NO:3). In someembodiments the motif comprises a ‘G’ rather than ‘P’ at position 2,e.g., LGXTG (SEQ ID NO:13), e.g., LGATG (SEQ ID NO:14). In someembodiments the motif comprises an ‘I’ rather than ‘L’ at position 1,e.g., IPXTG (SEQ ID NO:15), e.g., IPNTG (SEQ ID NO:16) or IPETG (SEQ IDNO: 17). Additional suitable sortase recognition motifs will be apparentto those of skill in the art, and the invention is not limited in thisrespect. It will be appreciated that the terms “recognition motif” and“recognition sequence”, with respect to sequences recognized by atransamidase or sortase, are used interchangeably.

In some embodiments of the invention the sortase is a sortase B (SrtB),e.g., a sortase B of S. aureus, B. anthracis, or L. monocytogenes.Motifs recognized by sortases of the B class (SrtB) often fall withinthe consensus sequences NPXTX, e.g., NP[Q/K]-[T/s]-[N/G/s], such asNPQTN (SEQ ID NO:4) or NPKTG (SEQ ID NO:5). For example, sortase B of S.aureus or B. anthracis cleaves the NPQTN (SEQ ID NO:4) or NPKTG (SEQ IDNO:5) motif of IsdC in the respective bacteria (see, e.g., Marraffini,L. and Schneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436,2007). Other recognition motifs found in putative substrates of class Bsortases are NSKTA (SEQ ID NO:18), NPQTG (SEQ ID NO:19), NAKTN (SEQ IDNO:20), and NPQSS (SEQ ID NO:21). For example, SrtB from L.monocytogenes recognizes certain motifs lacking P at position 2 and/orlacking Q or K at position 3, such as NAKTN (SEQ ID NO:22) and NPQSS(SEQ ID NO:23) (Mariscotti J F, Garcia-Del Portillo F, Pucciarelli MG.The Listeria monocytogenes sortase-B recognizes varied amino acids atposition two of the sorting motif. J Biol Chem. 2009 Jan. 7.)

In some embodiments, the sortase is a sortase C (Srt C). Sortase C mayutilize LPXTX as a recognition motif, with each occurrence of Xindependently representing any amino acid residue.

In some embodiments, the sortase is a sortase D (Srt D). Sortases inthis class are predicted to recognize motifs with a consensus sequenceNA-[E/A/S/H]-TG (Comfort D, supra). Sortase D has been found, e.g., inStreptomyces spp., Corynebacterium spp., Tropheryma whipplei,Thermobifida fusca, and Bifidobacterium longhum. LPXTA (SEQ ID NO:24) orLAXTG (SEQ ID NO:25) may serve as a recognition sequence for sortase D,e.g., of subfamilies 4 and 5, respectively subfamily-4 and subfamily-5enzymes process the motifs LPXTA (SEQ ID NO:26) and LAXTG (SEQ IDNO:27), respectively). For example, B. anthracis Sortase C has beenshown to specifically cleave the LPNTA (SEQ ID NO:28) motif in B.anthracis BasI and BasH (see Marrafini, supra).

See Barnett and Scott for description of a sortase that recognizesQVPTGV (SEQ ID NO:29) motif (Barnett, T C and Scott, J R, DifferentialRecognition of Surface Proteins in Streptococcus pyogenes by Two SortaseGene Homologs. Journal of Bacteriology, Vol. 184, No. 8, p. 2181-2191,2002; the entire contents of which are incorporated herein byreference). Additional sortases, including, but not limited to, sortasesand sortase variants recognizing additional sortase recognition motifsare also suitable for use in some embodiments of this invention. Forexample, sortases described in Chen I. et al., “A general strategy forthe evolution of bond-forming enzymes using yeast display.” Proc NatlAcad Sci USA. 2011 Jul. 12; 108(28):11399; Dorr, B. M., et al.,“Reprogramming the specificity of sortase enzymes.” Proc. Natl. Acad.Sci. U.S.A. 2014, 111, 13343-13348; the entire contents of each of whichare incorporated herein by reference.

In some embodiments, a variant of a naturally occurring sortase may beused. Such variants may be produced through processes such as directedevolution, site-specific modification, etc. Considerable structuralinformation regarding sortase enzymes, e.g., sortase A enzymes, isavailable, including NMR or crystal structures of SrtA alone or bound toa sortase recognition sequence (see, e.g., Zong Y, et al. J. Biol Chem.2004, 279, 31383-31389). Three dimensional structure information is alsoavailable for other sortases, e.g., S. pyogenes SrtA (Race, P R, et al.,J Biol Chem. 2009, 284(11):6924-33). The active site and substratebinding pocket of S. aureus SrtA have been identified. One of ordinaryskill in the art can generate functional variants by, for example,avoiding deletions or substitutions that would disrupt or substantiallyalter the active site or substrate binding pocket of a sortase. In someembodiments a functional variant of S. aureus SrtA comprises His atposition 120, Cys at position 184, and Arg at position 197, wherein Cysat position 184 is located within a TLXTC motif. Functional variants ofother SrtA proteins may have His, Cys, Arg, and TLXTC motifs atpositions that correspond to the positions of these residues in S.aureus SrtA. In some embodiments, a sortase variant comprises a sequenceat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a wild type sortase A sequence or catalytic domain thereof,e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to amino acids 60-206 of SEQ ID NO: 87 or SEQ ID NO: 88,or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to amino acids 26-206 of SEQ ID NO: 87 or SEQ ID NO: 88.In some embodiments, a sortase variant comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to aminoacids 60-206 of SEQ ID NO: 87 or relative to amino acids 26-206 of SEQID NO: 87 or SEQ ID NO: 88.

In some embodiments, a transamidase having higher transamidase activitythan a naturally occurring sortase may be used. In some embodiments theactivity of the transamidase is at least about 10, 15, 20, 40, 60, 80,100, 120, 140, 160, 180, or 200 times as high as that of S. aureussortase A. In some embodiments the activity is between about 10 and 50times as high as that of S. aureus sortase A, e.g., between about 10 and20 times as high, between about 20 and 30 times as high, between about30 and 50 times as high. In some embodiments the activity is betweenabout 50 and about 150 times as high as that of S. aureus sortase A,e.g., between about 50 and 75 times as high, between about 75 and 100times as high, between about 100-125 times as high, or between about 125and 150 times as high. For example, variants of S. aureus sortase A withup to a 140-fold increase in LPETG-coupling activity compared with thestarting wild-type enzyme have been identified (Chen, I., et al., PNAS108(28): 11399-11404, 2011). In some embodiments such a sortase variantis used in a composition or method of the invention. In some embodimentsa sortase variant comprises any one or more of the followingsubstitutions relative to a wild type S. aureus SrtA: P94S or P94R,D160N, D165A, K190E, and K196T mutations.

One of ordinary skill in the art will appreciate that the foregoingdescriptions of substitutions utilize standard notation of the formX₁NX₂, in which X₁ and X₂, represent amino acids and N represents anamino acid position, X₁ represents an amino acid present in a firstsequence (e.g., a wild type S. aureus SrtA sequence), and X₂ representsan amino acid that is substituted for X₁ at position N, resulting in asecond sequence that has X₂ at position N instead of X₁. It should beunderstood that the present disclosure is not intended to be limited inany way by the identity of the original amino acid residue X₁ that ispresent at a particular position N in a wild type SrtA sequence used togenerate a SrtA variant and is replaced by X₂ in the variant. Anysubstitution which results in the specified amino acid residue at aposition specified herein is contemplated by the disclosure. Thus asubstitution may be defined by the position and the identity of X2,whereas X₁ may vary depending, e.g., on the particular bacterial speciesor strain from which a particular SrtA originates. Thus in someembodiments, a sortase A variant comprises any one or more of thefollowing: an S residue at position 94 (S94) or an R residue at position94 (R94), an N residue at position 160 (N160), an A residue at position165 (A165), an E residue at position 190 (E190), a T residue at position196 (T196) (numbered according to the numbering of a wild type SrtA,e.g., SEQ ID NO: 87). For example, in some embodiments a sortase Avariant comprises two, three, four, or five of the afore-mentionedmutations relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 87).In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94), and also an Nresidue at position 160 (N160), an A residue at position 165 (A165), anda T residue at position 196 (T196). For example, in some embodiments asortase A variant comprises P94S or P94R, and also D160N, D165A, andK196T. In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94) and also an Nresidue at position 160 (N160), A residue at position 165 (A165), a Eresidue at position 190, and a T residue at position 196. For example,in some embodiments a sortase A variant comprises P94S or P94R, and alsoD160N, D165A, K190E, and K196T. In some embodiments a sortase A variantcomprises an R residue at position 94 (R94), an N residue at position160 (N160), a A residue at position 165 (A165), E residue at position190, and a T residue at position 196. In some embodiments a sortasecomprises P94R, D160N, D165A, K190E, and K196T.

It is to be further understood that the disclosure contemplates variantsof any wild-type sortase A. Those skilled in the art will appreciatethat wild-type sequences of sortase A may vary, e.g., SrtA from variousspecies may have gaps, insertions, and/or may vary in length relative tothe amino acid sequence of exemplary wild-type S. aureus SrtA. Thoseskilled in the art will appreciate that the positions described hereinin regard to substitutions or other alterations pertain to the sequenceof exemplary wild type S. aureus SrtA, unless otherwise indicated, andthat such positions may be adjusted when making correspondingsubstitutions in different bacterial SrtA sequences in order to accountfor such gaps, insertions, and/or length differences. For example, asnoted above, certain sortase variants comprise a substitution at aminoacid position 94 (e.g., the amino acid is changed to an S residue).However, the amino acid at position 94 in S. aureus SrtA may correspondto an amino acid at a different position (e.g., position Z) in SrtA froma second bacterial species when the sequences are aligned. Whengenerating a variant of the SrtA of the second bacterial speciescomprising a substitution at “position 94” (based on the wild type S.aureus SrtA sequence numbering), it is the amino acid at position Z ofthe SrtA from the second bacterial species that should be changed (e.g.,to S) rather than the amino acid at position 94. Those skilled in theart will understand how to align any original wild-type sortase Asequence to be used for generating a SrtA variant with an exemplarywild-type S. aureus sortase A sequence for purposes of determining thepositions in the original wild-type sortase A sequence that correspondto the exemplary wild-type S. aureus sortase A sequence when taking intoaccount gaps and/or insertions in the alignment of the two sequences.

In some embodiments, amino acids at position 94, 160, 165, 190, and/or196 are altered in a variant as compared with the amino acids present atthose positions in a wild type S. aureus SrtA, and the other amino acidsof the variant are identical to those present at the correspondingpositions in a wild type SrtA, e.g., a wild type S. aureus SrtA. In someembodiments, one or more of the other amino acids of a variant, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 of the other amino acids differ from thosepresent at corresponding position(s) in a wild type SrtA, e.g., a wildtype S. aureus SrtA. In some embodiments a variant may have any of theproperties or degrees of sequence identity specified in the definitionof “variants” above.

An exemplary wild type S. aureus SrtA sequence (Gene ID: 1125243, NCBIRefSeq Acc. No. NP_375640.1) is shown below, with the afore-mentionedpositions underlined:

(SEQ ID NO: 87) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDNKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK.One of ordinary skill in the art will appreciate that differentsubspecies, strains, and isolates may differ in sequence at positionsthat do not significantly affect activity. For example, anotherexemplary wild type S. aureus SrtA sequence (Gene ID: 3238307, NCBIRefSeq Acc. No. YP_187332.1; GenBank Acc. No. AAD48437) has a K residueat position 57 and a G residue at position 167. as shown below in SEO IDNO: 88:

(SEQ ID NO: 88) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK

Either or both of these amino acids (i.e., K57 and/or G167) may bepresent in or introduced into any SrtA sequence, e.g., any S. aureusSrtA sequence, whether naturally occurring or generated by man.Furthermore, as described herein, any sortase sequence may furthercomprise a tag (e.g., 6×His), a spacer, or both. For example, the N- orC-terminus may be extended to encompass a tag, optionally separated fromthe rest of the sequence by a spacer,

In some embodiments a sortase variant comprising the following sequencemay be used, in which amino acid substitutions relative to a wild typeS. aureus SrtA of SEQ ID NO: 87 or SEQ ID NO: 88 are shown in underlinedbold letters:

(SEQ ID NO: 89) MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPAT R EQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI R N VKPT AVEVLDEQKGKDKQLTLITCDDYNE E TGVWE T RKIFVATEVK.

As will be appreciated, amino acids 2-148 of the above sequencecorrespond to amino acids 60-206 of the full length S. aureus SrtAsequence (the catalytic domain). For example, the “R” residue atposition 36 of SEQ ID NO: 89 corresponds to the “P” residue at position94 in SEQ ID NO: 87 or 88. It is also contemplated in some embodimentsto use sortase variants that have other substitutions at one or more ofpositions 94, 160, 165, 190, and 196 (numbered according to thenumbering of SEQ ID NO: 87 or 88), e.g., wherein such substitutionsutilize an amino acid that would be a conservative substitution at therelevant position as compared with the sequence of SEQ ID NO: 89.

The use of sortases found in any gram-positive organism, such as thosementioned herein and/or in the references (including databases) citedherein is contemplated in the context of some embodiments of thisinvention. Also contemplated is the use of sortases found in gramnegative bacteria, e.g., Colwellia psychrerythraea, Microbulbiferdegradans, Bradyrhizobium japonicum, Shewanella oneidensis, andShewanella putrefaciens. Such sortases recognize sequence motifs outsidethe LPXTX consensus, for example, LP[Q/K]T[A/S]T. In keeping with thevariation tolerated at position 3 in sortases from gram-positiveorganisms, a sequence motif LPXT[A/S], e.g., LPXTA (SEQ ID NO:30) orLPSTS (SEQ ID NO:31) may be used.

Those of skill in the art will appreciate that any sortase recognitionmotif known in the art can be used in some embodiments of thisinvention, and that the invention is not limited in this respect. Forexample, in some embodiments the sortase recognition motif is selectedfrom: LPKTG (SEQ ID NO:32), LPITG (SEQ ID NO:33), LPDTA (SEQ ID NO:34),SPKTG (SEQ ID NO:35), LAETG (SEQ ID NO:36), LAATG (SEQ ID NO:37), LAHTG(SEQ ID NO:38), LASTG (SEQ ID NO:39), LAETG (SEQ ID NO:40), LPLTG (SEQID NO:41), LSRTG (SEQ ID NO:42), LPETG (SEQ ID NO:2), VPDTG (SEQ IDNO:43), IPQTG (SEQ ID NO:44), YPRRG (SEQ ID NO:45), LPMTG (SEQ IDNO:46), LPLTG (SEQ ID NO:47), LAFTG (SEQ ID NO:48), LPQTS (SEQ IDNO:49), it being understood that in various embodiments of the inventionthe fifth residue may be replaced with any other amino acid residue. Forexample, the sequence used may be LPXT, LAXT, LPXA, LGXT, IPXT, NPXT,NPQS (SEQ ID NO:50), LPST (SEQ ID NO:51), NSKT (SEQ ID NO:52), NPQT (SEQID NO:53), NAKT (SEQ ID NO:54), LPIT (SEQ ID NO:55), LAET (SEQ IDNO:56), or NPQS (SEQ ID NO:57). The invention encompasses embodiments inwhich ‘X’ in any sortase recognition motif disclosed herein or known inthe art is amino acid, for example, any naturally occurring or anynon-naturally occurring amino acid. In some embodiments, X is selectedfrom the 20 standard amino acids found most commonly in proteins foundin living organisms. In some embodiments, e.g., where the recognitionmotif is LPXTG (SEQ ID NO:1) or LPXT, X is D, E, A, N, Q, K, or R. Insome embodiments, X in a particular recognition motif is selected fromthose amino acids that occur naturally at position 3 in a naturallyoccurring sortase substrate. For example, in some embodiments X isselected from K, E, N, Q, A in an LPXTG (SEQ ID NO:1) or LPXT motifwhere the sortase is a sortase A. In some embodiments X is selected fromK, S, E, L, A, N in an LPXTG (SEQ ID NO:1) or LPXT motif and a class Csortase is used.

In some embodiments, a sortase recognition sequence further comprisesone or more additional amino acids, e.g., at the N- or C-terminus. Forexample, one or more amino acids (e.g., up to five amino acids) havingthe identity of amino acids found immediately N-terminal to, orC-terminal to, a five amino acid recognition sequence in a naturallyoccurring sortase substrate may be incorporated. Such additional aminoacids may provide context that improves the recognition of therecognition motif.

The term “sortase substrate,” as used herein, refers to any moleculethat is recognized by a sortase, for example, any molecule that canpartake in a sortase-mediated transpeptidation reaction. In someembodiments, “sortase substrate” and “sortase substrate peptide” areused interchangeably. A typical sortase-mediated transpeptidationreaction involves a substrate comprising a C-terminal sortaserecognition motif, e.g., an LPXTX motif, and a second substratecomprising an N-terminal sortase recognition motif, e.g., an N-terminalpolyglycine or polyalanine. In some embodiments, a sortase recognitionmotif, though described as being “C-terminal” or N-terminal,” is notrequired to be at the immediate C- or N-terminus. For example, in someembodiments, other amino acids, for example a tag (e.g., a 6×His-tag),are found at the immediate C-terminus of a protein comprising aC-terminal sortase recognition motif, and the C-terminal sortaserecognition motif is adjacent (e.g., within 5, 10, 15 or 20 amino acids)thereto. A sortase substrate may be a peptide or a protein, for example,a peptide comprising a sortase recognition motif such as an LPXTX motifor a polyglycine or polyalanine, wherein the peptide is conjugated to anagent, e.g., a radiolabeled compound or small molecule. Accordingly,both proteins and non-protein molecules can be sortase substrates aslong as they comprise a sortase recognition motif. Some examples ofsortase substrates are described in more detail elsewhere herein andadditional suitable sortase substrates will be apparent to the skilledartisan. The invention is not limited in this respect.

The term “sortagging,” as used herein, refers to the process of adding atag or agent, e.g., a moiety or molecule, for example, a radiolabeledcompound or small molecule, onto a target molecule, for example, atarget protein for use in PET applications via a sortase-mediatedtranspeptidation reaction. Examples of additional suitable tags include,but are not limited to, amino acids, nucleic acids, polynucleotides,sugars, carbohydrates, polymers, lipids, fatty acids, and smallmolecules. Other suitable tags will be apparent to those of skill in theart and the invention is not limited in this aspect. In someembodiments, a tag comprises a sequence useful for purifying,expressing, solubilizing, and/or detecting a polypeptide. In someembodiments, a tag can serve multiple functions. In some embodiments, atag comprises an HA, TAP, Myc, 6×His, Flag, streptavidin, biotin, or GSTtag, to name a few examples. In some embodiments, a tag is cleavable, sothat it can be removed, e.g., by a protease. In some embodiments, thisis achieved by including a protease cleavage site in the tag, e.g.,adjacent or linked to a functional portion of the tag. Exemplaryproteases include, e.g., thrombin, TEV protease, Factor Xa, PreScissionprotease, etc. In some embodiments, a “self-cleaving” tag is used. See,e.g., Wood et al., International PCT Application PCT/US2005/05763, filedon Feb. 24, 2005, and published as WO/2005/086654 on Sep. 22, 2005.

The term “subject” includes, but is not limited to, vertebrates, morespecifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a birdor a reptile or an amphibian. In some embodiments, the subject is ahuman subject. As used herein, “patient” refers to a subject afflictedwith a disease or disorder. The term “patient” includes human andveterinary subjects.

The term “target protein,” as used herein in the context ofsortase-mediated modification of proteins, refers to a protein that ismodified by the conjugation of an agent, for example a radioactive agentthat renders the protein suitable for diagnostic and/or therapeuticapplications such as PET. The term “target protein” may refer to a wildtype or naturally occurring form of the respective protein, or to anengineered form, for example, to a recombinant protein variantcomprising a sortase recognition motif not contained in a wild-type formof the protein. The term “modifying a target protein,” as used herein inthe context of sortase-mediated protein modification, refers to aprocess of altering a target protein comprising a sortase recognitionmotif via a sortase-mediated transpeptidation reaction. Typically, themodification leads to the target protein being conjugated to an agent,for example, a peptide, protein, detectable label, or small molecule. Incertain embodiments, the modification provides radiolabeled proteins.

The terms “thiosemicarbazide” or “thiosemicarbazide group,” are usedinterchangeably herein, and refer to functional groups having thegeneral formula:

wherein R⁴⁷ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R⁴⁷ is an optionally substituted amino acid. In certain embodiments, theamino acid is within a peptide chain.

The term “thiosemicarbazone,” as used herein, refers to compound havingthe general formula:

wherein each of R⁴⁷, R⁴⁸, and R⁴⁹ is independently optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl In some embodiments, at least one ofR⁴⁷, R⁴⁸, and R⁴⁹ is an optionally substituted amino acid. The term“thiosemicarbazone linkage,” as used herein, refers to the formula:

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Site-specific enzymatic labeling of proteins has emerged as a generalmethod for derivatizing proteins with various types of modifications.For example, the transpeptidation reaction catalyzed by sortases can beused for various types of modifications. For conventional sortasemodifications, target proteins are engineered to include a sortaserecognition motif (e.g., LPXTG; SEQ ID NO:1) near their C-termini. Whenincubated with peptides containing one or more N-terminal glycineresidues and a sortase, these sortase substrates undergo atransacylation reaction resulting in the exchange of residues C-terminalto the threonine residue in the case of the LPXTG recognition sequencewith the oligoglycine peptide, resulting in the protein C-terminus beingligated to the N-terminus of the peptide.

This invention is based, at least in part, on the recognition thatsortases can be exploited to conjugate a variety of moieties todiagnostic and/or therapeutic proteins. Such sortase-mediatedconjugation (e.g., “sortagging”) approaches can be used to confer newfunctions to diagnostic/therapeutic proteins. For example, theconjugation of a radioactive agent (e.g., a radiolabel) to a desiredprotein that binds to specific tissues or cells (e.g., an antibody)allows for the use of such in positron emission tomography (PET),single-photon emission computed tomography (SPECT), or combined imagingmethods such as PET/CT (PET with concurrent computed tomography imaging)or PET/MRI (PET with concurrent magnetic resonance imaging). For anotherexample, sortase-mediated conjugation of radionuclides to therapeuticproteins or antibodies may further provide tumoricidal effects, e.g., inthe treatment of cancer.

Some aspects of this disclosure provide methods, reagents, compositions,systems, and kits that can be used to modify proteins, for example, byconjugating such proteins to an agent or molecule comprising aradionuclide. Also provided are reagents and techniques which allow forthe conjugation of any agent or molecule comprising a carbonyl group(e.g., an aldehyde or ketone) or a click chemistry handle to a sortasepeptide substrate. Such modified substrates, in turn, can be conjugatedto a protein of interest using the sortase-mediated transpeptidationtechniques described herein. Notably, many commercially availablereagents can be employed using the methodology provided herein, whichfurther provides for efficiently and reproducibly radiolabeling proteinsof interest.

¹⁸F-fludeoxyglucose (FDG) is an analog of glucose wherein theradioactive isotope fluorine-18 (¹⁸F) is substituted for the typicalhydroxyl group at the 2′ position of glucose. The uptake of ¹⁸F-FDG bytissues is a marker for the tissue uptake of glucose, which in turn isclosely correlated with certain types of tissue metabolism. After¹⁸F-FDG is injected into a patient, a PET scanner can form twodimensional or three dimensional images of the distribution of ¹⁸F-FDGwithin the body. The images can be assessed by a nuclear medicinephysician or radiologist to provide diagnoses of various medicalconditions. Because of the wide use and availability of FDG, methods andreagents provided herein were developed, in part, to utilize FDG as aready source of ¹⁸F for radiolabeling proteins of interest, e.g.,proteins suitable for PET diagnostic/therapeutic applications.

However, any (radioactive) molecule or agent can be utilized in thepresent invention. For example, as described in Example 2, anotherreadily available source of ¹⁸F, ¹⁸F sodium fluoride (¹⁸F—NaF) can beused, in a substitution reaction, with sortagging technology and clickchemistry reactions such as a tetrazine (Tz)/trans-cyclooctene (TCO)reverse-electron demand Diels-Alder cycloaddition reaction to modifyproteins (e.g., antibodies). As another example, described in Example 3,a tetrazine-aminooxy molecule can be reacted with ¹⁸F-FDG to generate an¹⁸F-labeled click chemistry handle. This radiolabeled click chemistryhandle may be used to label a pre-prepared sortagged protein (e.g.,VHH-TCO) to form a final ¹⁸F-labeled protein.

In some embodiments, the molecule or agent comprises a carbonyl group ora click chemistry handle. Molecules and agents that include a carbonylgroup also include those that can form a carbonyl group throughisomerization. For example, reducing sugars, such as glucose and FDG,can isomerize to form a linear molecule having an aldehyde group, andare thus amenable for use in the methods provided herein.

Certain aspects of the present invention make use ofelectrophile-nucleophile pairs to generate sortase substrate peptidescomprising desired modifications, such as the incorporation of aradiolabel. These sortase substrate peptides, can then be used to modifyproteins of interest using the sortase-mediated transpeptidation methodsdescribed herein. For example, carbonyl groups (e.g., aldehydes andketones) react with aminooxy groups, hydrazides, and thiosemicarbazidesto form oximes, hydrazones, and thiosemicarbazones, respectively. Thus,modifying agents (e.g., radioactive agents such as FDG, ¹⁴C—(U)-glucose,etc.) that include a carbonyl can be chemically joined to sortasesubstrate peptides that comprise an aminooxy functional group,hydrazide, or thiosemicarbazide. Without wishing to be bound by anyparticular theory, it is believed that the alpha effect renders thenucleophilic nitrogen atoms of aminooxy groups, hydrazides, andthiosemicarbazides less basic but more nucleophilic than an amino group.Because of this enhanced nucleophilicity, these reactions arethermodynamically favorable in water. When ligation reactions areperformed at mildly acidic pH (e.g., 4.0-5.5) enhanced selectivity isobserved since the attenuated basicity of aminooxy groups, hydrazides,and thiosemicarbazides leaves these nucleophiles unprotonated whilepotentially competing amino groups are protonated (and therefore are notnucleophilic). Oxime, hydrazone, and thiosemicarbazone linkages arestable from pH 5 to pH 7, and have been used extensively inchemoselective ligation applications including e.g., the formation ofpeptide conjugates, in vivo protein labeling, and the generation ofenzyme inhibitors (See, e.g., Rashidian et al., “A highly efficientcatalyst for oxime ligation and hydrazone-oxime exchange suitable forbioconjugation.” Bioconjug. Chem. 2013 Mar. 20; 24(3):333-42; Langenhanet al., “Recent Carbohydrate-Based Chemoselective LigationApplications.” Current Organic Synthesis. 2005; 2, 59-81; the entirecontents of each are incorporated herein by reference).

In another embodiment, a sortase substrate peptide is first tethered toa protein of interest using a sortase-mediated transpeptidation reactionand subsequently modified to incorporate a label (e.g., a radiolabel,such as ¹⁸F). For example, a sortase substrate peptide comprising aclick-chemistry handle may be tethered to a protein of interest using asortase-mediated transpeptidation reaction. The protein of interest,containing the complementary click chemistry handle, is then labeled(e.g., with a radiolabel) using any suitable click chemistry reactionknown in the art.

Another means of modifying proteins and peptide substrates for use inthe methods described herein makes use of the tetrazine(Tz)/trans-cyclooctene (TCO) reverse-electron demand Diels-Aldercycloaddition reaction (See, e.g., Example 2, FIG. 4). The TCO-tetrazinereaction is the fastest known bioorthogonal reaction to date, with anestimated second order rate constant of 2000±400 M⁻¹s⁻¹ (Blackman etal., “The Tetrazine Ligation: Fast Bioconjugation based onInverse-electron-demand Diels-Alder Reactivity.” J. Am. Chem. Soc. 2008;130, 13518-13519). Additionally, use of catalysts, as described herein,allow for the quick formation of these linkages, for example, in about 5minutes, in about 10 minutes, in about 25 minutes, or in about 30minutes. Once modified, these sortase substrate peptides may be used ina sortagging reaction which conjugates the substrate peptide to adesired protein (e.g., comprising a complementary sortase recognitionmotif), for example, in about 10 minutes, in about 25 minutes, in about30 minutes or in about 45 minutes. Alternatively sortase substratepeptides may be modified following the sortagging reaction. Thesereaction times are amenable to the use of ¹⁸F-labeled, or otherradioactive agents, and therefore represent a novel, fast, and facilemethod to generate radiolabeled proteins for use in diagnostic and/ortherapeutic applications such as PET.

Radiolabled Proteins

Another aspect of the present invention provides radiolabeled proteins.The radiolabeled proteins can be prepared from modified proteins ofFormula (I).

In certain embodiments, provided herein is a modified protein of Formula(I):

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences; and

R¹ comprises a reactive group capable of undergoing a click chemistryreaction.

As generally defined herein, L¹ is a linker formed by enzymaticconjugation between two enzyme recognition sequences. In certainembodiments, L¹ comprises at least four amino acids. In certainembodiments, L¹ comprises at least five amino acids. In certainembodiments, L¹ comprises at least six amino acids. In certainembodiments, L¹ comprises at least seven amino acids. In certainembodiments, L¹ is a linker formed by sortase-mediated transpeptidationof two sortase recognition sequences. In certain embodiments, L¹ is-LPXTGGGK-, -LPXTGGG-, -NPXTGGGK-, NPXTGGG-, -LPXTAAA-, -NPXTAAA-,-LPXTGGGGG-, or -LPGAG-, wherein each instance of X is independently anamino acid. In certain embodiments, X is E. In certain embodiments, X isQ. In certain embodiments, X is K.

In certain embodiments, the modified protein is formed by enzymaticconjugation of

and a compound of Formula (a): B—R¹ (a), wherein each of A and B isindependently an enzyme recognition sequence. In certain embodiments,the modified protein is formed by sortase-mediated transpeptidation of

and the compound of Formula (a): B—R¹ (a), wherein A comprises aC-terminal sortase recognition sequence, and B comprises a N-terminalsortase recognition sequence; or A comprises a N-terminal sortaserecognition sequence, and B comprises a C-terminal sortase recognitionsequence.

In certain embodiments, A comprises LPXTX or NPXTX, and B comprises anoligoglycine or an oligoalanine sequence; wherein each instance of X isindependently an amino acid. In certain embodiments, B comprises LPXTXor NPXTX, and A comprises an oligoglycine or an oligoalanine sequence;wherein each instance of X is independently an amino acid. In certainembodiments, A is LPETG (SEQ ID NO:2), LPETA (SEQ ID NO:3), NPQTN (SEQID NO:4), or NPKTG (SEQ ID NO:5), and B is GGG or AAA. In certainembodiments, A comprises an oligoglycine or an oligoalanine sequence,and B comprises LPXTX or NPXTX, wherein each instance of X isindependently an amino acid. In certain embodiments, B is LPETG (SEQ IDNO:2), LPETA (SEQ ID NO:3), NPQTN (SEQ ID NO:4), or NPKTG (SEQ ID NO:5),and A is GGG or AAA.

As used herein, the enzyme recognition sequence is an amino acidsequence recognized by a transamidase enzyme. In certain embodiments,the transamidase recognition sequence is a sortase recognition sequenceor a sortase recognition motif. In certain embodiments, the sortase issortase A (SrtA). In certain embodiments, the sortase is sortase B(SrtB).

As generally defined herein, R¹ is a reactive group capable ofundergoing a click chemistry reaction.

Click chemistry is a chemical approach introduced by Sharpless in 2001and describes chemistry tailored to generate substances quickly andreliably by joining small units together. See, e.g., Kolb, Finn andSharpless Angewandte Chemie International Edition (2001) 40: 2004-2021;Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplarycoupling reactions (some of which may be classified as “Clickchemistry”) include, but are not limited to, formation of esters,thioesters, amides (e.g., such as peptide coupling) from activated acidsor acyl halides; nucleophilic displacement reactions (e.g., such asnucleophilic displacement of a halide or ring opening of strained ringsystems); azide-alkyne Huisgon cycloaddition; thiol-yne addition; imineformation; Michael additions (e.g., maleimide addition); Diels-Alderreaction and inverse electron demand Diels-Alder reaction; and [4+1]cycloadditions (e.g. between isonitriles (isocyanides) and tetrazines).In certain embodiments, the click chemistry reaction is a Diels-Alderreaction. It is to be understood that the click chemistry reaction maybe followed by additional one or more chemical transformations. Incertain embodiments, the click chemistry reaction is a Diels-Alderreaction followed by an retro-Diels-Alder reaction.

In certain embodiments, R¹ is a reactive group capable of undergoing a[3+2] cycloaddition. In certain embodiments, R¹ comprises adipolarophile. In certain embodiments, R¹ comprises an alkynyl group. Incertain embodiments, R¹ comprises comprises a 1,3-dipole. In certainembodiments, R¹ comprises an azido. In certain embodiments, R¹ is areactive group capable of undergoing a a Diels-Alder cycloaddition. Incertain embodiments, R¹ comprises a conjugated diene. In certainembodiments, R¹ comprises a tetrazine or a quadricyclane. In certainembodiments, R¹ comprises a tetrazine. In certain embodiments, R¹comprises an unsubstituted tetrazine. In certain embodiments, R¹comprises a substituted tetrazine.

In certain embodiments, R¹ is of Formula (i):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; and

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene.

As generally defined herein, R^(t) is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, or optionallysubstituted heterocyclyl. In certain embodiments, R^(t) is hydrogen. Incertain embodiments, R^(t) is optionally substituted aliphatic. Incertain embodiments, R^(t) is optionally substituted C₁₋₆ alkyl. Incertain embodiments, R^(t) is unsubstituted C₁₋₆ alkyl. In certainembodiments, R^(t) is methyl or ethyl. In certain embodiments, R^(t) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(t) is optionallysubstituted aryl. In certain embodiments, R^(t) is optionallysubstituted phenyl. In certain embodiments, R^(t) is optionallysubstituted heteroaryl. In certain embodiments, R^(t) is optionallysubstituted pyridine.

As generally defined herein, R^(s) is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, R^(s) is a bond. Incertain embodiments, R^(s) is optionally substituted aliphatic. Incertain embodiments, R^(s) is optionally substituted C₁₋₆ alkyl. Incertain embodiments, R^(s) is unsubstituted C₁₋₆ alkyl. In certainembodiments, R^(s) is methyl or ethyl. In certain embodiments, R^(s) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(s) is optionallysubstituted heteroaliphatic. In certain embodiments, R^(s) is optionallysubstituted arylene. In certain embodiments, R^(s) is optionallysubstituted phenyl. In certain embodiments, R^(s) is optionallysubstituted heteroarylene. In certain embodiments, R^(s) is optionallysubstituted pyridine.

In certain embodiments, R¹ comprises a dienophile. In certainembodiments, R¹ comprises an optionally substituted alkene. In certainembodiments, R¹ comprises a cyclooctene. In certain embodiments, R¹comprises a substituted cyclooctene of the formula:

wherein R^(A1) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene. R¹comprises a substituted cyclooctene of the formula:

In certain embodiments, R¹ comprises a trans-cyclooctene. In certainembodiments, R¹ comprises a substituted trans-cyclooctene of the formula

wherein R^(A1) is as defined herein. In certain embodiments, R¹comprises a substituted trans-cyclooctene of the formula

wherein R^(A1) is as defined herein. In certain embodiments, R¹comprises an unsubstituted trans-cyclooctene.

Another aspect of the invention provides a radioactive protein ofFormula (II)

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences; and

L² is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene.

In certain embodiments, the linker L² is formed by a click chemistryreaction. In certain embodiments, the linker L² is formed by a [3+2]cycloaddition. In certain embodiments, the linker L² is formed by aDiels-Alder cycloaddition. In certain embodiments, the linker L² isformed by a Diels-Alder cycloaddition followed by one or more chemicaltransformations. In certain embodiments, the linker L² is formed by aDiels-Alder cycloaddition followed by retro-Diels-Alder reaction.

As generally defined herein, L² is optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted arylene,optionally substituted heteroarylene, or optionally substitutedheterocyclylene. In certain embodiments, L² is optionally substitutedaliphatic. In certain embodiments, L² is optionally substitutedheteroaliphatic. In certain embodiments, L² is optionally substitutedarylene. In certain embodiments, L² is optionally substitutedcycloalkylene. In certain embodiments, L² is optionally substitutedheteroarylene.

In certain embodiments, the radioactive protein of Formula (II) isformed by a click chemistry reaction of the modified protein of Formula(I) and a compound of Formula (b): ¹⁸F—R² (b), wherein R² is a reactivegroup capable of undergoing the click chemistry reaction.

As generally defined herein, R² is a reactive group capable ofundergoing a click chemistry reaction. In certain embodiments, R² is areactive unsaturated group capable of undergoing a [3+2] cycloaddition.In certain embodiments, R² comprises a dipolarophile. In certainembodiments, R² comprises an alkynyl group. In certain embodiments, R²comprises a 1,3-dipole. In certain embodiments, R² comprises an azido.In certain embodiments, R² is a reactive group capable of undergoing a aDiels-Alder cycloaddition. In certain embodiments, R² comprises aconjugated diene. In certain embodiments, R² comprises a tetrazine or aquadricyclane. In certain embodiments, R² comprises a tetrazine. Incertain embodiments, R² comprises an unsubstituted tetrazine. In certainembodiments, R² comprises a substituted tetrazine.

In certain embodiments, R² is of Formula (i):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; and

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene.

In certain embodiments, R² comprises a dienophile. In certainembodiments, R² comprises an optionally substituted alkene. In certainembodiments, R² comprises a cyclooctene. In certain embodiments, R²comprises a trans-cyclooctene. In certain embodiments, R² comprises asubstituted trans-cyclooctene. In certain embodiments, R² comprises anunsubstituted trans-cyclooctene.

In certain embodiments, a compound of Formula (b): ¹⁸F—R² (b), is ofFormula (b-1):

wherein R^(A2) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; andR^(A2) comprises ¹⁸F. In certain embodiments, R^(A2) is optionallysubstituted aliphatic. In certain embodiments, R^(A2) is optionallysubstituted heteroaliphatic. In certain embodiments, R^(A2) is —O—C₁₋₆alkylene, wherein the C₁₋₆ alkylene comprises ¹⁸F.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-a):

wherein h is an integer of 1 to 5, inclusive.

Compounds of Formula (b-1-a) can be prepared from a compound of Formula(b-1-a-i):

with a proper nucleophile comprising ¹⁸F, wherein h is as definedherein, LG is a leaving group. In certain embodiments, the compounds ofFormula (b-1) can be prepared according to Scheme S6 or Scheme S6-a.

The term “leaving group” is given its ordinary meaning in the art ofsynthetic organic chemistry and refers to an atom or a group capable ofbeing displaced by a nucleophile. Examples of suitable leaving groupsinclude, but are not limited to, halogen (such as F, Cl, Br, or I(iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy,arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy,aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, and haloformates. Insome cases, the leaving group is a sulfonic acid ester, such astoluenesulfonate (tosylate, -OTs), methanesulfonate (mesylate, -OMs),p-bromobenzenesulfonyloxy (brosylate, -OBs), ortrifluoromethanesulfonate (triflate, -OTf). In some cases, the leavinggroup is a brosylate, such as p-bromobenzenesulfonyloxy. In some cases,the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. Insome embodiments, the leaving group is a sulfonate-containing group. Insome embodiments, the leaving group is a tosylate group. The leavinggroup may also be a phosphineoxide (e.g., formed during a Mitsunobureaction) or an internal leaving group such as an epoxide or cyclicsulfate. Other non-limiting examples of leaving groups are water,ammonia, alcohols, ether moieties, thioether moieties, zinc halides,magnesium moieties, diazonium salts, and copper moieties. In certainembodiments, LG is -OTs.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-b):

wherein L⁴ and Y are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-c):

wherein L⁴, M, L^(a), L^(b), and L^(c) are as defined herein, and - - -indicates a coordination bond or absent, as valency permits. In certainembodiments, - - - is a single coordination bond. In certainembodiments, - - - is absent.

In certain embodiments, a compound of Formula (b) is of the formula:

In certain embodiments, a compound of Formula (b): ¹⁸F R² (b), is ofFormula (b-2):

wherein R^(s) and R^(t) are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-2-a):

wherein R^(t), R^(p), and p are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a1):

wherein R^(t), R^(N1), L³, R^(q1), and q1 are as defined herein; andR^(G) is an optionally substituted carbohydrate group; provided thatR^(G) comprises ¹⁸F.

A “carbohydrate group” or a “carbohydrate” refers to a monosaccharide ora polysaccharide (e.g., a disaccharide or oligosaccharide). Exemplarymonosaccharides include, but are not limited to, natural sugars, such asallose, altrose, glucose, mannose, gulose, idose, galactose, talose,ribose, arabinose, xylose, and lyxose. Disaccharides are two joinedmonosaccharides. Exemplary disaccharides include, but are not limitedto, sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and ten monosaccharide units(e.g., raffinose, stachyose). The carbohydrate group may be a naturalsugar or a modified sugar. Exemplary modified sugars include, but arenot limited to, sugars where the hydroxyl group is replaced with anamino group and/or alkyl group (e.g., such as desosamine),2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibosewherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, or a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose), and the like. Variouscarbohydrates are further described below and herein. Carbohydrates mayexist in many different forms, for example, conformers, cyclic forms,acyclic forms, stereoisomers, tautomers, anomers, and isomers. Incertain embodiments, R^(G) is an optionally substituted glucose.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a2):

wherein R^(t), R^(N1) are as defined herein; and R^(G1) is an optionallysubstituted carbohydrate group or a fragment thereof; provided thatR^(G1) comprises ¹⁸F.

The oxime compounds of Formula (b-2-a2)

can be prepared from optionally substituted tetrazine-aminooxy and aradiolabeled optionally substituted aldehyde of the formula R^(as)-CHO,wherein R^(as) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl (SchemeS1). In certain embodiments, R^(as) is an optionally substitutedcarbohydrate group or a fragment thereof. In certain embodiments, R^(as)is an optionally substituted glucose or a fragment thereof. In certainembodiments, the reaction is carried out in the presence of a catalyst.In certain embodiments, the catalyst is m-phenylenediamine,p-phenylenediamine, or p-anisidine. In certain embodiments, the catalystis m-phenylenediamine. In certain embodiments, the molar ratio of theoptionally substituted tetrazine-aminooxy to the catalyst is from about10:1 to 1:10. In certain embodiments, the molar ratio of the optionallysubstituted tetrazine-aminooxy to the catalyst is from about 1:1 to 1:8.In certain embodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:1 to 1:6. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:2 to 1:4. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is about 1:4.

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof, provided R^(as) comprises ¹⁸F. In certainembodiments, R^(as) is an optionally substituted glucose or a fragmentthereof. In certain embodiments, R^(as) is ¹⁸F-FDG of a fragmentthereof.

As provided in Scheme S1, the resulting oxime product can be easilypurified from the reaction mixture to the change in hydrophilicity.

In certain embodiments of Scheme S1, the excess of tetrazine-aminooxycan be captured by reacting with another water soluble carbohydrate. Incertain embodiments, the water soluble carbohydrate is glucosamine6-sulfate.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a2):

wherein R^(t), R^(N1), L³, R^(s1), R^(s2), R^(s3), and R^(s4) are asdefined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a3):

wherein R^(t), R^(N1), L³, R^(s1), R^(s2), R^(s3), and R^(s4) are asdefined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-2-b):

wherein R^(t), R^(N1), L³, R^(q1), and q1 are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-b1):

In certain embodiments, a compound of Formula (b) is of Formula (b-3):

wherein R^(t), L⁴, and Y are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-3-a):

wherein R^(t), L⁴, L^(a), L^(b), and L^(c) are as defined herein.

In certain embodiments, a compound of Formula (b) is of the followingformula:

In certain embodiments, the linker L² is of Formula (ii):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl;

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

each instance of R^(c1) is hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl; or optionally two R^(c1) taken with the intervening atomsto form an optionally substituted carbocyclyl or optionally substitutedheterocyclyl ring;

R^(c2) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

m is 0, 1, 2, 3, 4, 5, 6, 7, or 8, as valency permits;

a indicates point of attachment to L¹; and

b indicates point of attachment to ¹⁸F.

In certain embodiments, m is 0. In certain embodiments, m is 1. Incertain embodiments, m is 2. In certain embodiments, m is 3. In certainembodiments, m is 4.

As generally defined herein, each instance of R^(c1) is independentlyhydrogen, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In certainembodiments, R^(c1) is hydrogen. In certain embodiments, R^(c1) isoptionally substituted aliphatic. In certain embodiments, R^(c1) isoptionally substituted alkyl. In certain embodiments, R^(c1) isoptionally substituted heteroaliphatic. In certain embodiments, twoR^(c1) taken with the intervening atoms to form an optionallysubstituted carbocyclyl. In certain embodiments, two R^(c1) taken withthe intervening atoms to form an optionally substituted cyclopropyl.

As generally defined herein, R^(c2) is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, R^(c2) is a bond.In certain embodiments, R^(c2) is optionally substituted aliphatic. Incertain embodiments, R^(c2) is optionally substituted alkyl. In certainembodiments, R^(c2) is optionally substituted heteroaliphatic. Incertain embodiments, R^(c2) is optionally substituted alkoxy. In certainembodiments, R^(c2) is an optionally substituted amino group.

In certain embodiments, the linker L² is of Formula (ii-a):

wherein n is an integer between 1 and 8, inclusive.

In certain embodiments, n is 1. In certain embodiments, n is 2. Incertain embodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5. In certain embodiments, n is 6. In certainembodiments, n is 7. In certain embodiments, n is 8.

In certain embodiments, the linker L² is of Formula (ii-b):

wherein n is an integer between 1 and 8, inclusive.

In certain embodiments, the linker L² is of Formula (iii):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl;

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

each instance of R^(c1) is hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl;

R^(c2) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

m is 0, 1, 2, 3, 4, 5, 6, 7, or 8, as valency permits;

a indicates point of attachment to L¹; and

b indicates point of attachment to ¹⁸F.

In certain embodiments, the linker L² is of Formula (iii-a):

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b):

wherein

each instance of R^(p) is independently hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, hydroxyl, or optionally substituted amino;provided at least one R^(p) is not hydrogen and comprises F¹⁸; and

p is 1, 2, 3, 4, or 5.

In certain embodiments, p is 1. In certain embodiments, p is 2. Incertain embodiments, p is 3. In certain embodiments, p is 4. In certainembodiments, p is 3. In certain embodiments, p is 5.

As generally defined herein, R^(p) is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, hydroxyl, or optionally substituted amino;wherein at least one R^(p) is not hydrogen and comprises F¹⁸. In certainembodiments, R^(p) is hydrogen. In certain embodiments, R^(p) isoptionally substituted aliphatic. In certain embodiments, R^(p) isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R^(p) isunsubstituted C₁₋₆ alkyl. In certain embodiments, R^(p) is methyl orethyl. In certain embodiments, R^(p) is substituted C₁₋₆ alkyl. Incertain embodiments, R^(p) is optionally substituted aryl. In certainembodiments, R^(p) is optionally substituted phenyl. In certainembodiments, R^(p) is optionally substituted heteroaryl. In certainembodiments, R^(p) is optionally substituted pyridine. In certainembodiments, least one R^(p) is not hydrogen and comprises F¹⁸.

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b1):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

each of R^(s1), R^(s2), R^(s3), and R^(s4) is independently hydrogen,optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, or optionally substitutedheteroaryl, or an oxygen protecting group.

As generally defined herein, L³ is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, L³ is a bond. Incertain embodiments, L³ is optionally substituted aliphatic. In certainembodiments, L³ is optionally substituted heteroaliphatic. In certainembodiments, L³ comprises an oxime moiety. In certain embodiments, L³ isof the formula

wherein c indicates the point of attachment to —N—R^(N1)—; d indicatesthe point of attachment to —CH¹⁸F—; and u is 1, 2, 3, 4, or 5. Incertain embodiments, L³ is C═O.

In certain embodiments, R^(N1) is independently hydrogen. In certainembodiments, R^(N1) is optionally substituted aliphatic. In certainembodiments, R^(N1) is optionally substituted alkyl. In certainembodiments, R^(N1) is an amino protectin group.

In certain embodiments, R^(s1) is independently hydrogen. In certainembodiments, R^(s1) is optionally substituted aliphatic. In certainembodiments, R^(s1) is optionally substituted alkyl. In certainembodiments, R^(s1) is an oxygen protectin group. In certainembodiments, R^(s1) is acyl (e.g. acetyl).

In certain embodiments, R^(s2) is independently hydrogen. In certainembodiments, R^(s2) is optionally substituted aliphatic. In certainembodiments, R^(s2) is optionally substituted alkyl. In certainembodiments, R^(s2) is an oxygen protectin group. In certainembodiments, R^(s2) is acyl (e.g. acetyl).

In certain embodiments, R^(s3) is independently hydrogen. In certainembodiments, R^(s3) is optionally substituted aliphatic. In certainembodiments, R^(s3) is optionally substituted alkyl. In certainembodiments, R^(s3) is an oxygen protectin group. In certainembodiments, R^(s3) is acyl (e.g. acetyl).

In certain embodiments, R^(s4) is independently hydrogen. In certainembodiments, R^(s4) is optionally substituted aliphatic. In certainembodiments, R^(s4) is optionally substituted alkyl. In certainembodiments, R^(s4) is an oxygen protectin group. In certainembodiments, R^(s4) is acyl (e.g. acetyl).

In certain embodiments, R^(s1), R^(s2), R^(s3), and R^(s4) are allhydrogen.

In certain embodiments, R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) areall hydrogen.

In certain embodiments, R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) areall hydrogen; and R^(t) is optionally substituted aliphatic. In certainembodiments, R^(N1), R^(s1), R^(s2), R^(s3) and R^(s4) are all hydrogen;and R^(t) is optionally substituted C₁₋₆ alkyl. In certain embodiments,R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) are all hydrogen; and R^(t)is methyl or ethyl.

In certain embodiments, wherein -L²-F¹⁸ is of the formula

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b2):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group;

each instance of R^(q1) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, hydroxyl, or optionally substitutedamino; and

q1 is 0, 1, 2, 3, or 4.

In certain embodiments, R^(t) is optionally substituted aliphatic andR^(q1) is hydrogen. In certain embodiments, R^(t) is optionallysubstituted C₁₋₆ alkyl; L³ is

u is 1; and R^(q1) is hydrogen.

In certain embodiments, -L²-F¹⁸ is of the formula:

In certain embodiments. -L²-F¹⁸ is of Formula (iii-b3):

wherein

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

each instance of R^(q2) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heteroaryl, optionally substituted heterocyclyl,hydroxyl, or optionally substituted amino; and

q2 is 0, 1, 2, 3, or 4.

In certain embodiments, q2 is 0. In certain embodiments, q2 is 1. Incertain embodiments, q1 is 2. In certain embodiments, q2 is 3. Incertain embodiments, q2 is 4.

In certain embodiments, R^(q2) is hydrogen. In certain embodiments,R^(q2) is optionally substituted aliphatic. In certain embodiments,R^(q2) is optionally substituted C₁₋₆ alkyl.

In certain embodiments, R^(t) is optionally substituted aliphatic andR^(q2) is hydrogen.

In certain embodiments, -L²-F¹⁸ is of the formula:

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b4):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

R^(Z) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, wherein RZ comprise ¹⁸F.

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b4-a):

In certain embodiments, R^(z) is an optionally substituted thiol.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c):

wherein

Y is a ligand capable of chelating to a pharmaceutically acceptablemetal complex comprising F¹⁸; and

L⁴ is is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted cycloalkylene, optionally substituted heteroarylene, oroptionally substituted heterocyclylene.

As generally defined herein, Y is a ligand capable of chelating to apharmaceutically acceptable metal complex comprising F¹⁸. As usedherein, a ligand refers to an ion or molecule (functional group) thatbinds to a central metal atom to form a coordination complex. Thebonding between metal and ligand generally involves formal donation ofone or more of the ligand's electron pairs. The nature of metal-ligandbonding can range from covalent to ionic. Exemplary monodentate ligandsinclude, but are not limited to, CO, organonitriles (e.g., CH₃CN,CH₃CH₂CN), monosubstituted amines, disubstituted amines, trisubstitutedamines, heterocyclyls (e.g., pyridine, piperidine), dialkylcyanamides,triphenylphosphine oxide, THF, DMF, or NMF. Exemplary bidentate ligandsinclude, but are not limited to, 1,5-cyclooctadiene, norbornadiene,1,2-ethylenediamine, tetramethylethylenediamine, 1,2-dimethoxyethane,diglyme, or 2,5-dithiahexane. Exemplary tridentate ligands include, butare not limited to, conjugated cyclic triene (e.g., cycloheptatriene),conjugated acyclic triene, arenes (e.g., benzene, toluene, xylene,mesitylene, naphthalene), tetraazamacrocyles (e.g.,tetraazacyclododecane), polyamines (e.g., diethylenetriamine), andtrithiocylononane. In certain embodiments, the ligand is a polydentateligand. In certain embodiments, the ligand comprises1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), ortriazacyclononane-phosphinate (TRAP).

The phrase “pharmaceutically acceptable” means that the metal complex issuitable for administration to a subject. In certain embodiments, themetal complex is a halide metal complex. In certain embodiments, themetal is a pharmaceutically acceptable metal. In certain embodiments,the metal is IIA or IIIA group metal. In certain embodiments, the metalis an early transition metal. In certain embodiments, the metal is Al.

In certain embodiments, L⁴ is a bond. In certain embodiments, L⁴ is anoptionally substituted aliphatic. In certain embodiments, L⁴ is anoptionally substituted heteroaliphatic.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c1):

wherein

M is a pharmaceutically acceptable metal;

each of L^(a), L^(b), and L^(c) is independently optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted cycloalkylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;and

“- - - ” indicates a coordination bond or absend, as valency permits.

In certain embodiments, L^(a) is optionally substituted aliphatic. Incertain embodiments, L^(a) is optionally substituted heteroaliphatic. Incertain embodiments, L^(a) is optionally substituted heteroalkylene. Incertain embodiments, L^(a) is —(CH₂)₁₋₃—C(═O)O—, wherein the chelationpoint to M is O. In certain embodiments, L^(a) is —CH₂—C(═O)O—. Incertain embodiments, L^(a) is —(CH₂)₁₋₃—C(═O)OH, wherein the chelationpoint to M is O. In certain embodiments, L^(a) is —CH₂—C(═O)OH.

In certain embodiments, L^(b) is optionally substituted aliphatic. Incertain embodiments, L^(b) is optionally substituted heteroaliphatic. Incertain embodiments, L^(b) is optionally substituted heteroalkylene. Incertain embodiments, L^(b) is —(CH₂)₁₋₃—C(═O)O— wherein the chelationpoint to M is O. In certain embodiments, L^(b) is —CH₂—C(═O)O—. Incertain embodiments, L^(b) is —(CH₂)₁₋₃—C(═O)OH, wherein the chelationpoint to M is O. In certain embodiments, L^(b) is —CH₂—C(═O)OH.

In certain embodiments, L^(c) is optionally substituted aliphatic. Incertain embodiments, L^(c) is optionally substituted heteroaliphatic. Incertain embodiments, L^(c) is optionally substituted heteroalkylene. Incertain embodiments, L^(c) is —(CH₂)₁₋₃—C(═O)O— wherein the point ofattachment to M is O. In certain embodiments, L^(c) is —CH₂—C(═O)O—. Incertain embodiments, L^(c) is —(CH₂)₁₋₃—C(═O)OH, wherein chelation pointto M is O. In certain embodiments, L^(c) is —CH₂—C(═O)OH.

As generally defined herein, “- - - ” indicates the chelation formedbetween M and the ligand, as valency permits. In certain embodiments, Mforms one chelating bond with the ligand. In certain embodiments, Mforms two chelating bonds with the ligand. In certain embodiments, Mforms three chelating bonds with the ligand.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c2):

In certain embodiments, the radioactive protein is one of the followingformulae:

In certain embodiments, the linker L² is of one of the followingformulae:

wherein R^(s), R^(t), R^(c1), R^(c2), and m are as defined herein.

In certain embodiments, provided herein is a radioactive protein ofFormula (III)

wherein

L¹ is as defined herein; and

R³ comprises a ligand capable of chelating to a pharmaceuticallyacceptable radioactive metal complex.

In certain embodiments, L¹-R³ is of the following formula:

wherein R^(z2) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, or optionallysubstituted heteroaryl, or a nitrogen protecting group; wherein R^(z2)comprises a ligand capable of chelating to a pharmaceutically acceptableradioactive metal complex.

In certain embodiments, R³ comprises mono-dentecate ligand. In certainembodiments, R³ comprises a polydentecate ligand. In certainembodiments, R³ comprises 1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), ortriazacyclononane-phosphinate (TRAP).

In certain embodiments, the metal is ⁶⁴Cu²⁺. In certain embodiments, themetal is ⁶⁸Ga³⁺.

In certain embodiments, the radioactive protein is one of the followingformulae:

In certain embodiments, provided herein is a radioactive protein ofFormula (IV)

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences;

R⁴ comprises a radioactive optionally substituted carbohydrate; and

R⁴ is linked to the C-terminus of the adjacent amino acid in L¹.

As generally defined herein, R⁴ comprises a radioactive optionallysubstituted carbohydrate. In certain embodiments, R⁴ comprises aradioactive optionally substituted glucose. In certain embodiments, R⁴comprises a radioactive glucose comprising ¹⁸F. In certain embodiments,R⁴ comprises an optionally substituted glucose comprising ¹⁸F. Incertain embodiments, R⁴ is linked to the C-terminus of the adjacentamino acid in L¹. In certain embodiments, R⁴ is linked to the side chainof the adjacent amino acid in L¹.

In certain embodiments, R⁴ is of Formula (iv):

wherein

v is 1, 2, 3, 4, or 5; and

each of R^(s5), R^(s6), R^(s7), and R^(s8) is independently hydrogen,optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, or optionally substitutedheteroaryl, or an oxygen protecting group.

In certain embodiments, R^(s5) is independently hydrogen. In certainembodiments, R^(s5) is optionally substituted aliphatic. In certainembodiments, R^(s5) is optionally substituted alkyl. In certainembodiments, R^(s5) is an oxygen protectin group. In certainembodiments, R^(s5) is acyl (e.g. acetyl).

In certain embodiments, R^(s6) is independently hydrogen. In certainembodiments, R^(s6) is optionally substituted aliphatic. In certainembodiments, R^(s6) is optionally substituted alkyl. In certainembodiments, R^(s6) is an oxygen protectin group. In certainembodiments, R^(s6) is acyl (e.g. acetyl).

In certain embodiments, R^(s7) is independently hydrogen. In certainembodiments, R^(s7) is optionally substituted aliphatic. In certainembodiments, R^(s7) is optionally substituted alkyl. In certainembodiments, R^(s7) is an oxygen protectin group. In certainembodiments, R^(s7) is acyl (e.g. acetyl).

In certain embodiments, R^(s8) is independently hydrogen. In certainembodiments, R^(s8) is optionally substituted aliphatic. In certainembodiments, R^(s8) is optionally substituted alkyl. In certainembodiments, R^(s8) is an oxygen protectin group. In certainembodiments, R^(s8) is acyl (e.g. acetyl).

In certain embodiments, R⁴ is of the formula:

In certain embodiments, the radioactive protein is of the followingformula:

wherein R^(s5), R^(s6), R^(S7), and R^(s8) are as defined herein; andL^(G) is optionally substituted aliphatic or optionally substitutedheteroaliphatic.

In certain embodiments, L^(G) is optionally substituted aliphatic. Incertain embodiments, L^(G) is optionally substituted C₁₋₁₀ alkyl. Incertain embodiments, L^(G) is optionally substituted heteroaliphatic. Incertain embodiments, L^(G) is of the formula:

wherein e indicates the point of attachment to oxygen and f indicatesthe point of attachment to the alpha carbon of the amino acid.

In certain embodiments, the radioactive protein is of the followingformula:

In certain embodiments,

is an antibody, a nuclear factor, a neuropeptide, a receptor protein, anenzyme, a structural protein, or a fragment thereof. In certainembodiments,

is an antibody or a fragment thereof. In certain embodiments,

is VHH or a fragment thereof.

Synthesis of Intermediates and Radiolabeled Proteins

The oxime compounds of Formula (b-2-a2)

can be prepared from optionally substituted tetrazine-aminooxy and aradiolabeled optionally substituted aldehyde or optionally substitutedketone of the formula R^(as)—CO—R^(bs), wherein R^(G1) is as definedherein; each of R^(as) and R^(b)s is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl; provided R^(as) and R^(bs) are notboth hydrogen (Scheme S1).

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof. In certain embodiments, R^(as) is anoptionally substituted glucose or a fragment thereof. In certainembodiments, the reaction is carried out in the presence of a catalyst.In certain embodiments, the catalyst is m-phenylenediamine,p-phenylenediamine, or p-anisidine. In certain embodiments, the catalystis m-phenylenediamine. In certain embodiments, the molar ratio of theoptionally substituted tetrazine-aminooxy to the catalyst is from about10:1 to 1:10. In certain embodiments, the molar ratio of the optionallysubstituted tetrazine-aminooxy to the catalyst is from about 1:1 to 1:8.In certain embodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:1 to 1:6. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:2 to 1:4. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is about 1:4.

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof, provided R^(as) comprises ¹⁸F. In certainembodiments, R^(as) is an optionally substituted glucose or a fragmentthereof. In certain embodiments, R^(as) is ¹⁸F-FDG of a fragmentthereof.

As provided in Scheme S1, the resulting oxime product can be easilypurified from the reaction mixture to the change in hydrophilicity.

In certain embodiments of Scheme S1, the excess of tetrazine-aminooxycan be captured by reacting with another water soluble carbohydrate. Incertain embodiments, the water soluble carbohydrate is glucosamine6-sulfate.

The compound of Formula (b-2-b)

can be prepared from reacting an optionally substituted tetrazinecomprising a nucleophic group with an electrophile comprising ¹⁸F suchas ¹⁸F-SFB. Exemplary synthesis of Formula (b-2-b) is provided in SchemeS2.

In certain embodiments, the Nu is an amino group. In certainembodiments, the electrophile is an optionally substitutedN-succinimidyl comprising ¹⁸F. In certain embodiments, the optionallysubstituted N-succinimidyl is ¹⁸F-SFB of the formula

Exemplary synthesis of ¹⁸F-SFB can be found in FIG. 11.

The radioactive protein of Formula (II) can be prepared from a modifiedprotein of Formula (I) with a compound of Formula (b): ¹⁸F-R² (b),wherein R² is a reactive group capable of undergoing the click chemistryreaction (Scheme S3):

In certain embodiments, R¹ is the first click chemistry handle and R² isthe second click chemistry handle. In certain embodiments, R² is thefirst click chemistry handle and R¹ is the second click chemistryhandle.

Click chemistry should be modular, wide in scope, give high chemicalyields, generate inoffensive byproducts, be stereospecific, bephysiologically stable, exhibit a large thermodynamic driving force(e.g., >84 kJ/mol to favor a reaction with a single reaction product),and/or have high atom economy. Several reactions have been identifiedwhich fit this concept:

(1) The Huisgen 1,3-dipolar cycloaddition (e.g., the Cu(I)-catalyzedstepwise variant, often referred to simply as the “click reaction”; see,e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064).Copper and ruthenium are the commonly used catalysts in the reaction.The use of copper as a catalyst results in the formation of1,4-regioisomer whereas ruthenium results in formation of the1,5-regioisomer;

(2) Other cycloaddition reactions, such as the Diels-Aldercycloaddition;

(3) Nucleophilic addition to small strained rings like epoxides andaziridines;

(4) Nucleophilic addition to activated carbonyl groups; and

(4) Addition reactions to carbon-carbon double or triple bonds.

In certain embodiments, the click chemistry is a Diels-Aldercycloaddition. Exemplary Diels-Alder cycloadditions can be found in U.S.Patent Publication No. 20130266512, which is incorporated by referenceherein;

The radioactive protein of Formula (III) can be prepared from a compoundcomprising an aminooxy moiety with an optionally substituted aldehyde(Scheme S4):

The radioactive protein of Formula (IV) can be prepared from a compoundcomprising an aminooxy moiety with an optionally substituted aldehyde oran optionally substituted ketone (Scheme S5), wherein L^(G) is asdefined herein.

In certain embodiments, the aldehyde is an optionally substitutedcarbohydrate comprising an aldehyde group or is capable of forming onethrough isomerism. In certain embodiments, the optionally substitutedaldehyde is an optionally substituted monosaccharide. In certainembodiments, the optionally substituted aldehyde is optionallysubstituted glucose, optionally substituted glyceraldehyde, oroptionally substituted galactose. In certain embodiments, the optionallysubstituted aldehyde is optionally substituted glucose.

In certain embodiments, the catalyst is m-phenylenediamine (mPDA),o-phenylenediamine, p-phenylenediamine, o-aminophenol, m-aminophenol,p-aminophenol, o-aminobenzoic acid, 5-methoxyanthranilic acid,3,5-diaminobenzoic acid or aniline. In certain embodiments, the catalystis m-phenylenediamine (mPDA).

In certain embodiments, the radioactive optionally substitutedcyclooctene is synthesized by a nucleophilic reaction with an ¹⁸F anionwith a substituted cyclooctene comprising a leaving group LG (SchemeS6), wherein LG is as defined herein and L⁶¹ is optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene.

In certain embodiments, L⁶¹ is optionally substituted hereoaliphatic. Incertain embodiments, L⁶¹ is straight chain heteroaliphatic. In certainembodiments, L⁶¹ is —O—C₁₋₈alkylene. In certain embodiments, L⁶¹ is—O—(CH₂)₁₋₈—.

In certain embodiments, the radioactive optionally substitutedcyclooctene is synthesized as shown in Scheme S6-a, wherein L⁶¹ is asdefined herein.

In certain embodiments, the ¹⁸F⁻ anion is from an inorganic saltcomprising ¹⁸F⁻ anion. In certain embodiments, the ¹⁸F⁻ anion is from ametal salt comprising ¹⁸F⁻ anion. In certain embodiments, the ¹⁸F⁻ anionis from IA, IIA, or IIIA metal fluoride. In certain embodiments, the¹⁸F⁻ anion is from transition metal complex comprising ¹⁸F⁻.

In certain embodiments, the enzymatic conjugation is a modificationusing a formylglycine generating enzyme (FGE). In certain embodiments,the protein is an antibody. In certain embodiments, the enzyme is FGE.In certain embodiments, the FGE recognition sequence is CXPXR. Incertain embodiments, the FGE recognition sequence is LCTPSRGSLFTGR. Incertain embodiments, the radioactive protein is prepared according toScheme E1.

It is to be understood that the —CHO group generated from the FGEmodification can undergo any suitable reaction to incorporate aradioactive label, for example, a click chemistry handle, a radioactivecarbohydrate, or a ligand capable of chelating to a pharmaceuticallyacceptable radioactive metal complex. Exemplary transformations such asreacting with hydrazine or hydroxylamine are shown in the Scheme E1.

In certain embodiments, the enzymatic conjugation is a modificationusing sialyltransferases. In certain embodiments, the protein is a cellsurface polypeptide. In certain embodiments, the protein is a glycan. Anexemplary sialylation is shown in Scheme E2, wherein R⁴ is as definedherein.

In certain embodiments, R⁴ comprises radioactive optionally substitutedglucose. In certain embodiments, R⁴ comprises ¹⁸F-FDG. In certainembodiments, R⁴ comprises radioactive optionally substituted aldolase.In certain embodiments, R⁴ comprises radioactive optionally substitutedmannose.

In certain embodiments, the enzymatic conjugation is a modificationusing phosphopantetheinyltransferases (PPTases). In certain embodiments,the protein is peptide carrier protein (PCP). In certain embodiments,the protein is acyl carrier protein (ACP). In certain embodiments, thePPT recognition sequence comprises a serine residue. In certainembodiments, the PPTase recognition sequence is DSLEFIASKLA,VLDSLEFIASKLA, or GSQDVLDSLEFIASKLA. In certain embodiments, thephosphopantetheinyltransferase is Sfp. An exemplary modification usingPPTase is shown in Scheme E3, wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing polypeptidyltransferases (OGTases). In certain embodiments, theprotein is nuclear pore protein. In certain embodiments, the OGTase isUDP-Glc-NAc. In certain embodiments, the OGTase recognition sequencecomprises a serine residue or threonine residue. Exemplary modificationsusing polypeptidyltransferases are shown in Scheme E4, wherein R^(F) isas defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing transglutaminase (TGases). In certain embodiments, the protein isan antibody. In certain embodiments, the TGase recognition sequencecomprises a glutamine (Q) residue. In certain embodiments, the TGaserecognition sequence comprises XXQXX. In certain embodiments, theprotein recognition sequence is GGGSLLQG, PNPQLPF, PKPQQFM, or GQQQLG.In certain embodiments, the protein recognition sequence comprises alysine (K) residue. In certain embodiments, the protein recognitionsequence is MRHKGS. An exemplary modification using TGases is shown inScheme E5, wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing protein farnesyltransferase (PFTase). In certain embodiments, theprotein is an antibody. In certain embodiments, the PFTase recognitionsequence comprises CaaX, wherein each instance of a is independently analiphatic amino acid and X is as defined herein. Exemplary modificationsusing PFTases are shown in Scheme E6, wherein R^(F) is as definedherein.

In certain embodiments, the enzymatic conjugation is a modificationusing biotin ligases. In certain embodiments, the protein is anantibody. In certain embodiments, the biotin ligase recognition sequencecomprises lysine (K). In certain embodiments, the biotin ligaserecognition sequence comprises GLNDIFEAQKIEWHE. In certain embodiments,the enzyme is E. coli biotin ligase, BirA. An exemplary modificationusing biotin ligases is shown in Scheme E7, wherein R^(F) is as definedherein.

In certain embodiments, the enzymatic conjugation is a modificationusing lipoic acid ligases (LplAs). In certain embodiments, the proteinis an antibody. In certain embodiments, the protein is a growth factorreceptor. In certain embodiments, the LplA recognition sequencecomprises GFEIDKVWYDLDA. In certain embodiments, the enzyme is E. coliLpl. An exemplary modification using LplAs is shown in Scheme E8,wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing N-myristoyltransferase (NMT). In certain embodiments, the proteinis an antibody. In certain embodiments, the protein is a tyrosinekinase. In certain embodiments, the protein is a HIV-1 matrix protein.In certain embodiments, the protein is a HIV Gag. In certainembodiments, the protein is an ADP-ribosylating factor. In certainembodiments, the NMT recognition sequence comprises GXXXS/T, wherein Xis any amino acid. An exemplary modification using NMT is shown inScheme E9, wherein R^(F) is as defined herein.

In certain embodiments, R^(F) is a reactive group capable of undergoinga click chemistry reaction. In certain embodiments, R^(F) is R¹ asdefined herein. In certain embodiments, R^(F) is optionally substitutedtetrazine. In certain embodiments, R^(F) is optionally substitutedtetrazine comprising ¹⁸F. In certain embodiments, R^(F) is optionallysubstituted tetrazine comprising ¹⁸F-FDG or a fragment thereof. Incertain embodiments, R^(F) is optionally substituted tetrazinecomprising ¹⁸F-SFB or a fragment thereof. In certain embodiments, R^(F)is optionally substituted cyclooctene. In certain embodiments, R^(F) isoptionally substituted trans-cyclooctene. In certain embodiments, R^(F)is optionally substituted trans-cyclooctene comprising ¹⁸F. In certainembodiments, R^(F) is comprises a ligand capable of chelating to apharmaceutically acceptable radioactive metal complex. In certainembodiments, R^(F) is R³ as defined herein. In certain embodiments,R^(F) comprises a ligand capable of chelating to a pharmaceuticallyacceptable metal complex comprising F¹⁸. In certain embodiments, R^(F)is Y as defined herein. In certain embodiments, R^(F) comprises aradioactive optionally substituted carbohydrate. In certain embodiments,R^(F) is R⁴ as defined herein. In certain embodiments, R^(F) comprises¹⁸F-FDG or a fragment thereof.

Methods and Reagents for Sortase-Mediated Radiolabeling of Proteins

The present invention provides methods, compositions, reagents, and kitsfor the modification or labeling of proteins and peptides usingsortase-mediated transpeptidation of sortase substrate peptides thathave been modified to include a desired modification, e.g., aradiolabel, or are isotopically enriched. Typically, a method oflabeling a protein as provided herein comprises conjugating the targetprotein with an agent or a click chemistry handle via a sortase-mediatedtranspeptidation reaction. In order for a sortase-mediatedtranspeptidation to be possible, both the target protein and the agentmust be recognized by the sortase and must be capable of acting as asubstrate of the sortase in the transpeptidation reaction. Accordingly,the methods for labeling of proteins provided herein involve targetproteins and agents that comprise or are conjugated to a sortaserecognition motif. Some proteins and some agents (e.g., peptidescomprising a radiolabel and/or a reactive moiety) may comprise asuitable sortase recognition motif. However, in some embodiments, thetarget protein and/or the agent is engineered to comprise a suitablesortase recognition motif, for example, via protein engineering (e.g.,using recombinant technologies) or via chemical synthesis (e.g., linkinga non-protein agent to a sortase recognition motif). Methods formodifying, engineering, or synthesizing proteins (e.g., to include asortase recognition motif and/or reactive moiety) are known, and includethose described by Green and Sambrook, Molecular Cloning: A LaboratoryManual (4th ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2012)), and Nilsson et al., “Chemical Synthesis ofProteins.” Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 91-118; theentire contents of each are hereby incorporated by reference.

It should be appreciated that enzymes other than sortase may be used forthe enzymatic modification of proteins as described herein. Exemplaryenzymatic modifications include, but are not limited to, modificationsby formylglycine generating enzyme (FGE), modifications bysialyltransferase, modifications by phosphopantetheinyltransferase(PPTases), O-GlcNAc modifications by polypeptidyltransferase (OGTases),modifications by transglutaminase (TGases), modifications by proteinfarnesyltransferase (PFTases), modifications by biotin ligases, andmodifications by lipoic acid ligase (Rashidian et al., BioconjugateChem. 2013, 24, 1277-1294). In certain embodiments, a transglutaminasemay be used to site-specifically incorporate an agent into a protein bycatalyzing an acyl transfer reaction between the carboxamide group of aglutamine residue and a variety of unbranched primary amines, commonlythe ε-amino group of lysine. As another example, a proteinfarnesyltransferase may be used to transfer a farnesyl group to thecysteine of a CaaX tetrapeptide sequence at the carboxyl terminus of aprotein, where C is cysteine, a is an aliphatic amino acid, and X is anyamino acid. In certain embodiments, a formylglycine generating enzyme(FGE) is used to modify any of the proteins described herein. FGErecognizes a pentapeptide consensus sequence, CxPxR (where x is anyamino acid), and site-specifically oxidizes the cysteine in thissequence to an aldehyde-bearing formylglyine. The FGE recognitionsequence, or aldehyde tag, can be inserted into heterologous recombinantproteins produced in either prokaryotic or eukaryotic expressionsystems. By introducing this motif into proteins and subsequentlyreacting it with FGE, an aldehyde group can be site-specifically addedto the protein, which can then be used for covalent modification usingcomplementary aminooxy- or hydrazide-functionalized reagents. Otherenzymes that may be used for the labeling of proteins include, but arenot limited to, phosphopantetheinyltransferase, biotin ligase, lipoicacid ligase and N-myristoyl transferase. Exemplary enzymes and theircorresponding recognition motifs are shown in table 3.

TABLE 3Kinetic parameters of different enzymes used for protein labelingk_(cat) K_(M) k_(cat)/K_(M) site of the enzyme encoded tag peptide min⁻¹μM min⁻¹/μM peptide Formylglycine CXPXR or 13-mer — — — C or N terminusgenerating enzyme LCTPSRGSLFTGR PhosphopantetheinylACP, PCP or ybbR tags (for 500 μM ybbR 13 mer C or N terminus ortransferase (11 mer: DSLEFIASKLA; and biotin-CoA) flexible loops13 mer: VLDSLEFIASKLA; 14.7 60.8 0.242 17 mer: GSQDVLDSLEFIASKLA)Sortase LPXTG 16.2 5,500 0.003 Any section Transglutaminase XXQXX 45 7 6Any section Famesyltransferase CaaX (for yPFTase and 2.4 μM C terminusCVIA) for FPP: 31.2 1.71 18.2 for FPP-aldehyde: 7.8 1.87 4.17Biotin ligase GLNDIFEAQKIEWHE (for Escherichia coli biotinC or N terminus ligase and biotin) 9.6 4.2 2.3 Lipoic acid ligaseGFEIDKVWYDLDA 2.88 — — C or N terminus or flexible loops

Typically, a method for protein labeling as provided herein comprisescontacting a target protein comprising a sortase recognition motif(e.g., a diagnostic protein comprising a C-terminal recognition motif),with an agent comprising a complementary sortase recognition motif(e.g., a sortase substrate peptide comprising an N-terminal recognitionmotif (e.g., GGG)), in the presence of a sortase under conditionssuitable for the sortase to conjugate the target protein to the agentvia a sortase-mediated transpeptidation reaction.

For example, some embodiments provide methods for labeling a targetprotein with a radioactive agent (e.g., a sortase substrate peptidecomprising a radioactive agent) by sortagging the agent to a diagnosticor therapeutic protein comprising a sortase recognition motif. Themethods include contacting the target protein with a sortase substratepeptide conjugated to an agent (e.g., a radioactive agent) in thepresence of a sortase under conditions suitable for the sortase toligate (e.g., transamidate) the sortase substrate to the target protein.In some embodiments, the target protein comprises a C-terminal sortaserecognition motif, and the sortase substrate peptide conjugated to theagent comprises an N-terminal sortase recognition motif. In otherembodiments, the target protein comprises an N-terminal sortaserecognition motif, and the sortase substrate peptide conjugated to theagent comprises a C-terminal sortase recognition motif. The C- andN-terminal recognition motif are recognized as substrates by the sortasebeing employed and ligated in a transpeptidation reaction.

In some embodiments, the methods involve contacting a target proteinwith a sortase substrate peptide conjugated to an agent (e.g., an agentcomprising a reactive moiety such as a click chemistry handle) in thepresence of a sortase under conditions suitable for the sortase totransamidate the sortase substrate peptide to the target protein.Following the transpeptidation reaction, the target protein is thenreacted with a complementary click chemistry handle which results inattaching a label (e.g., a radiolabel) to the target protein. Forexample, as described in Example 2, in some embodiments a target protein(e.g., an antibody) is sortagged with a sortase substrate peptidecomprising a reactive moiety (e.g., a tetrazine derivative clickchemistry handle), and the sortagged protein is then reacted with apartner reactive moiety (e.g., a trans-cyclooctene-containing moleculelabeled with ¹⁸F) to yield a radiolabeled protein. See, e.g., Example 2,FIG. 4.

Sortase-mediated transpeptidation reactions (also sometimes referred toas transacylation reactions) are catalyzed by the transamidase activityof sortase, which forms a peptide linkage (an amide linkage), between anacyl donor compound and a nucleophilic acyl acceptor containing anNH₂—CH₂-moiety. In some embodiments, the sortase employed to carry out asortase-mediated transpeptidation reaction is sortase A (SrtA). In someembodiments, the sortase employed to carry out a sortase-mediatedtranspeptidation reaction is sortase B (SrtB). However, it should benoted that any sortase, or transamidase, including engineered sortases,catalyzing a transacylation reaction can be used in the presentinvention, as the invention is not limited to the use of any particularsortase.

In certain embodiments, a sortase-mediated transpeptidation reaction forC-terminal modification or labeling of a protein, for example, of adiagnostic or therapeutic protein, is provided that comprises the stepof contacting a protein comprising a C-terminal sortase recognitionsequence of the structure:

-

-

wherein:

-   -   PRT is a target protein;    -   the sortase recognition motif is a C-terminal sortase        recognition motif, e.g., an LP(Xaa)TG (SEQ ID NO:1) motif,        wherein Xaa represents any amino acid residue;    -   X⁵¹ is —O—, —NR^(s51)—, or —S—; wherein R^(x51) is hydrogen,        substituted or unsubstituted aliphatic, or substituted or        unsubstituted heteroaliphatic;    -   R⁵¹ is H, acyl, substituted or unsubstituted aliphatic,        substituted or unsubstituted heteroaliphatic, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl;        with a nucleophilic moiety conjugated to an agent, according to        the formula:

-

wherein

-   -   the sortase recognition motif is an N-terminal sortase        recognition motif, for example, a polyglycine (G_(n1)) or        polyalanine (A_(n1)) motif (wherein n1 is an integer between        0-100 inclusive) or a (G)_(n1)K motif, wherein n1 is an integer        between 1 and 10, inclusive;    -   the agent is any molecule or compound comprising one or more        radionuclides, for example, an amino acid, a peptide, a protein,        a nucleotide, a polynucleotide, a carbohydrate (e.g., ¹⁸F-FDG,        ¹⁴C—(U)-glucose, etc.), a click chemistry handle, a tag, a metal        atom, a non-polypeptide polymer, a synthetic polymer, a small        molecule, a lipid, a compound, or a label;

in the presence of a sortase, under conditions suitable to form amodified protein of formula:

-

-

In certain embodiments, a sortase-mediated transpeptidation reaction forN-terminal modification or labeling of a protein, for example, of adiagnostic or therapeutic protein, is provided that comprises a step ofcontacting a protein comprising an N-terminal sortase recognitionsequence of the structure:

-

wherein:

-   -   PRT is a target protein;    -   the sortase recognition motif is an N-terminal sortase        recognition motif, for example, a polyglycine (G_(n2)) or        polyalanine (A_(n2)) motif (wherein n2 is an integer between        0-100 inclusive);        with an agent conjugated to a C-terminal sortase recognition        motif, of the formula:

-

-

wherein

-   -   the agent is any molecule or compound comprising one or more        radionuclides, for example, an amino acid, a peptide, a protein,        a nucleotide, a polynucleotide, a carbohydrate (e.g., ¹⁸F-FDG,        ¹⁴C—(U)-glucose, etc.), a click chemistry handle, a tag, a metal        atom, a non-polypeptide polymer, a synthetic polymer, a small        molecule, a lipid, a compound, or a label;    -   the sortase recognition motif is a C-terminal sortase        recognition motif, e.g., an LP(Xaa)TG motif (SEQ ID NO:1),        wherein Xaa represents any amino acid residue;    -   X⁶¹ is —O—, —NR^(x61)—, or —S—; wherein R^(x61) is hydrogen,        substituted or unsubstituted aliphatic, or substituted or        unsubstituted heteroaliphatic; and    -   R⁶¹ is H, acyl, substituted or unsubstituted aliphatic,        substituted or unsubstituted heteroaliphatic, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl;        in the presence of a sortase, under conditions suitable to form        a protein of formula:

-

-

.

Any C-terminal sortase recognition motif may be used in the presentinvention. The invention is not limited in this respect. The C-terminalsortase recognition motif need only be compatible with the sortase beingused. In some embodiments, the C-terminal sortase recognition motif isLPXT, wherein X is a standard or non-standard amino acid. In someembodiments, X is selected from D, E, A, N, Q, K, or R. In someembodiments, the recognition sequence is selected from LPXT, LPXT, SPXT,LAXT, LSXT, NPXT, VPXT, IPXT, and YPXR. In some embodiments, X isselected to match a naturally occurring transamidase recognitionsequence. In some embodiments, the transamidase recognition sequence isselected from LPKT (SEQ ID NO:58), LPIT (SEQ ID NO:59), LPDT (SEQ IDNO:60), SPKT (SEQ ID NO:61), LAET (SEQ ID NO:62), LAAT (SEQ ID NO:63),LAET (SEQ ID NO:64), LAST (SEQ ID NO:65), LAET (SEQ ID NO:66), LPLT (SEQID NO:67), LSRT (SEQ ID NO:68), LPET (SEQ ID NO:69), VPDT (SEQ IDNO:70), IPQT (SEQ ID NO:71), YPRR (SEQ ID NO:72), LPMT (SEQ ID NO: 73),LPLT (SEQ ID NO:74), LAFT (SEQ ID NO:75), LPQT (SEQ ID NO:76), NSKT (SEQID NO:77), NPQT (SEQ ID NO:78), NAKT (SEQ ID NO:79), and NPQS (SEQ IDNO:80). In some embodiments, e.g., in certain embodiments in whichsortase A is used, the transamidase recognition motif comprises theamino acid sequence X₁PX₂X₃G, where X₁ is leucine, isoleucine, valine,or methionine; X₂ is any amino acid; X₃ is threonine, serine, oralanine; P is proline and G is glycine. In some embodiments, theC-terminal glycine is omitted. In specific embodiments, as noted above,X₁ is leucine and X₃ is threonine. In certain embodiments, X₂ isaspartate, glutamate, alanine, glutamine, lysine, or methionine. Incertain embodiments, e.g., where sortase B is utilized, the recognitionsequence often comprises the amino acid sequence NPX₁TX₂, where X₁ isglutamine or lysine; X₂ is asparagine or glycine; N is asparagine; P isproline, and T is threonine. The invention encompasses the recognitionthat selection of X may be based at least in part in order to conferdesired properties on the compound containing the recognition motif. Insome embodiments, X is selected to modify a property of the compoundthat contains the recognition motif, such as to increase or decreasesolubility in a particular solvent. In some embodiments, X is selectedto be compatible with reaction conditions to be used in synthesizing acompound comprising the recognition motif, e.g., to be unreactivetowards reactants used in the synthesis. One of ordinary skill willappreciate that, in certain embodiments involving naturally-occurringC-terminal sortase recognition motifs or engineered motifs comprisingfive amino acids, the C-terminal amino acid of the C-terminal sortaserecognition motif may be omitted. For example, an acyl group, mayreplace the C-terminal amino acid of the sortase recognition motif. Insome embodiments, the acyl group is an ester. In some embodiments, theacyl group is

In some embodiments, the agent to be conjugated to the target protein isa protein. In some embodiments, the agent is a peptide. In someembodiments, the agent is a radionuclide. In some embodiments, the agentis a peptide comprising a radionuclide, e.g., a radiolabeled sortasesubstrate peptide. In some embodiments, the agent is isotopicallyenriched (e.g., the agent has been enriched in one particular isotope ofan element and depleted in other isotopic forms of the element). In someembodiments, the agent comprises a reactive moiety, such as a clickchemistry handle (e.g., tetrazine, tetrazine-aminooxy, tetrazinedienophiles, azide, tetrazoles, nitrile imines, trans-cyclooctene (TCO),difluorinated cyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone,cyclopropyl-fused bicyclononyne, norbornene, biscyclononene, andunactivated alkenes).

In certain embodiments, n2 (designating the number of amino acids in theN-terminal sortase recognition motif) is an integer from 0 to 50,inclusive. In certain embodiments, n2 is an integer from 0 to 20,inclusive. In certain embodiments, n2 is 0. In certain embodiments, n2is 1. In certain embodiments, n2 is n1, wherein n1 is an integer of 1 to10, inclusive. In certain embodiments, n1 is 2. In certain embodiments,n1 is 3. In certain embodiments, n1 is 4. In certain embodiments, n1 is5. In certain embodiments, n1 is 6.

Any sortase that can carry out a transpeptidation reaction underconditions suitable for conjugating e.g., a radiolabeled sortasesubstrate peptide to a target protein, can be used this invention.Examples of suitable sortases include, but are not limited to, sortase Aand sortase B, for example, from Staphylococcus aureus, or Streptococcuspyogenes. Additional sortases suitable for use in this invention will beapparent to those of skill in the art, including, but not limited to anyof the 61 sortases described in Dramsi S, Trieu-Cuot P, Bierne H,Sorting sortases: a nomenclature proposal for the various sortases ofGram-positive bacteria. Res Microbiol. 156(3):289-97, 2005, the entirecontents of which are incorporated herein by reference. Sortasesbelonging to any class of sortases, e.g., class A, class B, class C, andclass D sortases, and sortases belonging to any sub-family of sortases(subfamily 1, subfamily 2, subfamily 3, subfamily 4 and sub-family 5)can be used in this invention.

Any amino acid sequence recognized by a sortase can be used in thepresent invention. It will be understood by those of skill in the art,however, that in order for a certain sortase to carry out atranspeptidation reaction, the sortase recognition motif of the targetprotein to be modified and the sortase recognition motif the agent isconjugated to need to be recognized by that sortase. Numerous suitablesortase recognition motifs are provided herein, and additional suitablesortase recognition motifs will be apparent to the skilled artisan.Aside from naturally occurring sortase recognition motifs, someembodiments of this invention contemplate the use of non-naturallyoccurring sortase recognition motifs and sortases recognizing suchmotifs, for example, sortase motifs and sortases described in Piotukh etal., Directed evolution of sortase A mutants with altered substrateselectivity profiles. J Am Chem Soc. 2011 Nov. 9; 133(44):17536-9; andChen I, Dorr B M, and Liu D R. A general strategy for the evolution ofbond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011Jul. 12; 108(28):11399-404; the entire contents of each of which areincorporated herein by reference. In some embodiments, a recognitionsequence, e.g., a sortase recognition sequence as provided hereinfurther comprises one or more additional amino acids, e.g., at the N-and/or C-terminus. For example, one or more amino acids (e.g., up to 5amino acids) having the identity of amino acids found immediatelyN-terminal to, or C-terminal to, a five amino acid recognition sequencein a naturally occurring sortase substrate may be incorporated. Suchadditional amino acids may provide context that improves the recognitionof the recognition motif.

In some embodiments, suitable sortase recognition motifs may benaturally present in a target protein, for example, an N-terminal orC-terminal recognition motif sequence, in which case no furtherengineering of the target protein may be required. The skilled artisanwill understand that the choice of a suitable sortase for the(radio)labeling of a given target protein may depend on the sequence ofthe target protein, e.g., on whether or not the target protein comprisesa sequence at its C-terminus or its N-terminus that can be recognized asa substrate by any known sortase. In some embodiments, use of a sortasethat recognizes a naturally occurring C-terminal or N-terminalrecognition motif is preferred since further engineering of the targetprotein can be avoided.

Sortases, sortase-mediated transacylation reactions, and their use intranspeptidation (sometimes also referred to as transacylation) forprotein engineering are well known to those of skill in the art (see,e.g., Ploegh et al., International PCT Patent Application,PCT/US2010/000274, filed Feb. 1, 2010, published as WO 2010/087994 onAug. 5, 2010; Ploegh et al., International PCT Patent ApplicationPCT/US2011/033303, filed Apr. 20, 2011, published as WO 2011/133704 onOct. 27, 2011; and Ploegh et al., PCT/US2012/044584, filed Jun. 28,2012, published as WO 2013/003555 on Jan. 3, 2013; the entire contentsof each are incorporated herein by reference).

In some embodiments, methods for radiolabeling a protein having asortase recognition motif are provided. The methods comprise contactingthe protein with a sortase substrate peptide in the presence of asortase under conditions suitable for the sortase to transamidate theprotein and the sortase substrate peptide, wherein the sortase substratepeptide comprises a radiolabeled agent. In some embodiments, the sortasesubstrate peptide comprises a reactive moiety, such as a click chemistryhandle, which can be reacted with an agent comprising a complementaryclick chemistry handle and optionally radiolabel (e.g., ¹⁸F). In someembodiments, the radiolabeled agent is linked to the sortase substratepeptide by an oxime, a hydrazone, or a thiosemicarbazone, or through theuse of click chemistry, as described herein. Typically, the proteincomprises a C-terminal sortase recognition motif (as provided herein)and the sortase substrate peptide comprises an N-terminal sortaserecognition motif (as provided herein). However, as described above, insome embodiments the protein to be radiolabeled comprises an N-terminalsortase recognition motif and the sortase substrate peptide comprises aC-terminal sortase recognition motif.

Typically, the sortase substrate peptide comprises a radionuclide, or islinked (e.g., as described herein) to an agent (e.g., a small molecule)that comprises a radionuclide. The radionuclide is any radionuclidesuitable for use in diagnostic and/or therapeutic applications, forexample PET. Such radionuclides (isotopes) include, but are not limitedto, those radionuclides with positron emission (e.g., beta plus decay),such as carbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18,rubidium-82, copper-61, copper-62, copper-64, yttrium-86, gallium-68,zirconium-89, or iodine-124. In some embodiments, because of itsfavorable half-life (˜110 minutes), agents comprising fluorine-18 (¹⁸F)are conjugated to a sortase substrate peptide, for use in labeling atarget protein. For example, fludeoxyglucose (FDG, or ¹⁸F-FDG) is aradioactive analog of glucose that is the most prevalentradiopharmaceutical used in PET. Because of its wide-spread use andavailability, it is especially suited to the methods and uses describedherein. Similarly, sodium fluoride having ¹⁸F (¹⁸F—NaF) is widelyavailable and can be used as a source of ¹⁸F for labeling proteins andsortase substrate peptides with ¹⁸F (e.g., using substitution reactionsknown in the art). Thus, in some embodiments, proteins are labeled usingsortagging technology that makes use of sortase substrate peptideslinked to FDG or comprising ¹⁸F, as described herein. In someembodiments, the sortase substrate peptides are linked to¹⁴C—(U)-glucose. However, those of skill in the art will understand thatany agent comprising a suitable radionuclide (e.g., for use indiagnostic and/or therapeutic applications) can be linked to a sortasesubstrate peptide and used to label a target protein, as describedherein, and the invention is not limited in this respect.

Because of the half-lives of certain isotopes (e.g., ¹⁸F), the methods,compositions, reagents, and kits of the instant invention provide fastand efficient means to generate radiolabeled proteins. For example, insome embodiments, a target protein is labeled (e.g., sortagged with aradioactive sortase substrate peptide) in less than 5 minutes, less than10 minutes, less than 15 minutes, less than 20 minutes, less than 25minutes, less than 30 minutes, less than 45 minutes, less than 60minutes, less than 90 minutes, or less than 120 minutes. Additionally,in some embodiments, the methods, compositions, reagents, and kitsprovided herein allow for an efficient labeling reaction, wherein atleast 50%, at least 75%, at least 90%, at least 95%, or at least 98% ofthe protein is labeled with the agent. Methods for determining theefficiency (e.g., the amount of labeled protein compared to un-labeledprotein) of the labeling reaction are known, and include liquidchromatography-mass spectrometry (LC-MS). See, e.g., Lee et al., “LC/MSapplications in drug development.” Mass Spectrometry Reviews. 1999; 18(3-4): 187-279 and Wysocki et al., “Mass spectrometry of peptides andproteins.” Methods. 2005; 35 (3): 211-22; the entire contents of eachare hereby incorporated by reference.

In some embodiments, the methods described herein generate an amount ofpurified, radiolabeled protein having a suitable amount of radioactivityfor use in therapeutic and/or diagnostic applications, such as PET. Forexample, in some embodiments, the methods produce an amount ofradiolabeled protein comprising at least 10, at least 20, at least 30,at least 40, at least 50, at least 75, at least 100, at least 150, atleast 200, at least 250, at least 300, at least 350, at least 400, atleast 450, at least 500, at least 550, at least 600, at least 700, atleast 800, at least 900, or at least 1000 MBq of radioactivity.

In some embodiments, the protein to be labeled is a protein that isuseful in diagnostic and/or therapeutic applications, e.g., as describedherein. In some embodiments, the protein is an antibody, an affibody, asingle-domain antibody, a Fab fragment, or a therapeutic peptide. Insome embodiments, the protein comprises a VHH domain (e.g., VHH4, VHH7).

In some embodiments, the protein binds to a tumor cell, atumor-associated cell (e.g., neovasculature cell), or a tumor antigen.In other embodiments, the protein binds to any immune cell. Someexamples of immune cells include T-cells, B-cells, plasma cells,macrophages, dendritic cells, neutrophils, eosinophils, or mast cells.In some embodiments, the protein binds to a marker of inflammation. Forexample, in some embodiments, the protein is an antibody useful indiagnostic applications involving PET. Use of antibodies for PET basedapplications is referred to as immunoPET (See, e.g., Knowles et al.,“Advances in immuno-positron emission tomography: antibodies formolecular imaging in oncology.” J Clin Oncol. 2012; 30:3884-3892; theentire contents of which are hereby incorporated by reference). Suchantibodies include monoclonal antibodies known to target or bindcancerous cells or tissues in a subject's body. For example, anon-limiting list of antibodies approved by the U.S. Food and DrugAdministration (FDA) and the European Medicines Agency is provided inTable 4 of Salsano and Treglia, “PET imaging using radiolabeledantibodies: future direction in tumor diagnosis and correlateapplications.” Research and Reports in Nuclear Medicine. 2013: 3; 9-17,the entire contents of which are hereby incorporated by reference. Thetable is reproduced below.

TABLE 4 List of monoclonal antibodies approved by the US Food and DrugAdministration and the European Medicines Agency in cancer therapy.Brand Target: Approval year Antibody name Type antibody type ApplicationCompany EU USA Rituximab Rituxan, Chimeric IgG1 CD20 Non-HodgkinGenentech 1998 1997 MabThera lymphoma Trastuzumab Herceptin HumanizedIgG1 HER2 Beast cancer Genentech/Roche 2000 1998 Gemtuzumab Mylotarg*Humanized IgG4, CD33 Acute myeloid Wyeth/Pfizer NA 2000 ozogamicinimmunotoxin leukemia Alemtuzumab MabCampath, Humanized IgG1 CD52 Chronicmyeloid Genzyme 2001 2001 Campath-IH leukemia Ibritumomab Zevalin MurineIgG1 CD20 Non-Hodgkin Biogen Idec 2004 2002 tiuxecan lymphomaTositumomab Bexxar Murine IgG2a CD20 Non-Hodgkin Corixa/GSK NA 2003lymphoma Cecuximab Erbitux Chimeric IgG1 EGFR Colorectal cancer,Imclone/Lilly 2004 2004 head and neck cancer Bevacizumab AvastinHumanized IgG1 VEGF Colorectal cancer, Genentech/Roche 2005 2004non-small cell lung cancer Panitumumab Vectibix Human IgG2 EGFRColorectal cancer Amgen 2007 2006 Ofatumumab Arzerra Human IgG1 CD20Chronic lymphocytic Genmab 2010 2009 leukemia Denosumab Prolia HumanIgG2 RANK ligand Bone metastases, Amgen 2010 2010 giant cell tumor ofbone Ipilimumab Yervoy Human IgG1 CTLA-4 Melanoma BMS 2011 2011Brentuximab Adcetris Chimeric IgG1, CD30 Anaplastic large SeattleGenetics 2012 2011 vedotin drug-conjugate cell lymphoma, Hodgkinlymphoma Pertuzumab Perjeta Humanized IgG1 HER2 Breast cancerGenentech/Roche 2013 2012 Ado-trastuzumab Kadeyla Humanized IgG1, HER2Breast cancer Genentech/Roche in review 2013 emtansine drug-conjugateNote: *withdrawn in 2010. Abbreviations: CTLA 4, cytotoxic T-lymphocyteantigen 4; EGFR, epidermal growth factor receptor; HER, human epidermalreceptor; NA, not approved; VEGF, vascular endothelial growth factor.

Any of the antibodies disclosed in Table 4 of Salsano and Treglia can belabeled according to the methods provided herein. Other antibodiesamenable to labeling as described herein include, but are not limitedto, those described in Wright and Lapi, “Designing the magic bullet? Theadvancement of immuno-PET into clinical use.” J. Nucl. Med. 2013 August;54(8):1171-4; the entire contents of which are hereby incorporated byreference. These antibodies (see below) were successfully labeled withisotopes and were used in PET based diagnostic and/or therapeuticapplications. However, the antibodies were labeled via chemical meansthat are not always amenable to quickly generating labeled antibodieswith isotopes having a short half-life. Thus, such antibodies can bequickly and efficiently labeled with any desired isotope according tothe methods, compositions, reagents, and kits provided herein.Antibodies disclosed by Wright and Lapi, include:

Humanized A33 (huA33), which recognizes A33 antigen, which is known tobe expressed in greater than 95% of human colon adenocarcinomas. In astudy utilizing radiolabeled huA33 (Carrasquillo et al., “¹²⁴I-huA33antibody PET of colorectal cancer.” J. Nucl. Med. 2011; 52:1173-1180;the entire contents of which are hereby incorporated by reference), 25patients with primary or metastatic colorectal cancer (CRC) wereadministered 44.4-396 MBq (median, 343 MBq) of ¹²⁴I-huA33 with a totalof 10 mg of huA33. No adverse side effects were observed during thetreatment that could be attributed to the huA33. The antibody could beadministered via intravenous administration or hepatic arterial infusion(HAI), with HAI giving no detectable advantage over intravenousinjection. Eleven patients had 12 primary tumors, 10 of which weredetected via immuno-PET. Ten patients had liver metastases, all of whichwere detected by ¹²⁴I-huA33. Four of 7 patients with nodal metastasesdisplayed uptake of the ¹²⁴I-huA33, and 2 of 5 patients had lung lesionsthat were visualized by immuno-PET.

Radretumab (L19SIP), which targets an epitope contained in theextra-domain B of fibronectin, was labeled with ¹²⁴I and used toestablish provisional doses of ¹³¹I-labeled radretumab in 6 patientswith brain metastasis (Poli et al., “Radretumab radioimmunotherapy inpatients with brain metastasis: a ¹²⁴I-L19SIP dosimetric PET study.Cancer Immunol Res. 2013:OF1-OF10; the entire contents of which arehereby incorporated by reference).

Girentuximab (cG250), a chimeric antibody that binds carbonic anhydraseIX (CAIX), expressed in >95% of clear cell renal carcinoma (ccRCC), waslabeled with ¹²⁴I and used to detect such cancers (Divgi et al.,“Positron emission tomography/computed tomography identification ofclear cell renal cell carcinoma: results from the REDECT Trial.” J.Clin. Oncol. 2013; 31:187-194; the entire contents of which are herebyincorporated by reference).

Panitumumab, a fully humanized antibody that binds epidermal growthfactor receptor (EGFR), was successfully labeled with ⁸⁹Zr and used toimage colorectal tumor xenografts (Nayak et al., “PET and MR imaging ofmetastatic peritoneal and pulmonary colorectal cancer in mice with humanepidermal growth factor receptor 1-targeted ⁸⁹Zr-Labeled panitumumab.”J. Nucl. Med. 2012; 53:113-120; Chang et al., “Development andcharacterization of ⁸⁹Zr-labeled panitumumab for immuno-positronemission tomographic imaging of the epidermal growth factor receptor.”Mol. Imaging. 2013; 12:17-27; the entire contents of each are herebyincorporated by reference).

U36, a chimeric antibody that recognizes the v6 region of CD44, waslabeled with ⁸⁹Zr to image head and neck squamous cell carcinoma(Börjesson et al. “Radiation dosimetry of ⁸⁹Zr-labeled chimericmonoclonal antibody U36 as used for immuno-PET in head and neck cancerpatients.” J. Nucl. Med. 2009; 50:1828-1836; the entire contents ofwhich are hereby incorporated by reference).

Trastuzumab, cetuximab, and bevacizumab (see Table 4 above), were alsosuccessfully labeled with ⁸⁹Zr and used in PET applications (Dijkers etal., “Biodistribution of ⁸⁹Zr-trastuzumab and PET imaging ofHER2-positive lesions in patients with metastatic breast cancer.” Clin.Pharmacol. Ther. 2010; 87:586-592;www.cancer.gov/clinicaltrials/search/results?protocolsearchid511815785.Accessed Jul. 15, 2013; the entire contents of each are herebyincorporated by reference).

In some embodiments, the protein (e.g., antibody) to be radiolabeledbinds to a tumor antigen. In general, a tumor antigen can be anyantigenic substance produced by tumor cells (e.g., tumorigenic cells, orin some embodiments tumor stromal cells, e.g., tumor-associated cellssuch as cancer-associated fibroblasts). In many embodiments, a tumorantigen is a molecule (or portion thereof) that is differentiallyexpressed by tumor cells as compared with non-tumor cells. In otherembodiments, a tumor antigen is expressed on the surface of the cell.Tumor antigens may include, e.g., proteins that are normally produced invery small quantities and are expressed in larger quantities by tumorcells, proteins that are normally produced only in certain stages ofdevelopment, proteins whose structure (e.g., sequence orpost-translational modification(s)) is modified due to a mutation intumor cells, or normal proteins that are (under normal conditions)sequestered from the immune system. Tumor antigens may be useful in,e.g., identifying or detecting tumor cells (e.g., for purposes ofdiagnosis and/or for purposes of monitoring subjects who have receivedtreatment for a tumor, e.g., to test for recurrence) and/or for purposesof targeting various agents (e.g., therapeutic agents) to tumor cells.For example, in some embodiments, a radiolabeled antibody is providedcomprising an antibody or antibody fragment that binds a tumor antigen,thereby allowing detection of the tumor in vivo, e.g., using PET. Insome embodiments, a tumor antigen is an expression product of a mutatedgene, e.g., an oncogene or mutated tumor suppressor gene, anoverexpressed or aberrantly expressed cellular protein, an antigenencoded by an oncogenic virus (e.g., HBV; HCV; herpesvirus familymembers such as EBV, KSV; papilloma virus, etc.), or an oncofetalantigen. Oncofetal antigens are normally produced in the early stages ofembryonic development and largely or completely disappear by the timethe immune system is fully developed. Examples are alphafetoprotein(AFP, found, e.g., in germ cell tumors and hepatocellular carcinoma) andcarcinoembryonic antigen (CEA, found, e.g., in bowel cancers andoccasionally in lung and breast cancers). Tyrosinase is an example of aprotein normally produced in very low quantities but whose production isgreatly increased in certain tumor cells (e.g., melanoma cells). Otherexemplary tumor antigens include, e.g., CA-125 (found, e.g., in ovariancancer); MUC-1 (found, e.g., in breast cancer); epithelial tumor antigen(found, e.g., in breast cancer); melanoma-associated antigen (MAGE;found, e.g., in malignant melanoma); and prostatic acid phosphatase(PAP, found in prostate cancer). In some embodiments, a tumor antigen isat least in part exposed at the cell surface of tumor cells. In someembodiments, a tumor antigen comprises an abnormally modifiedpolypeptide or lipid, e.g., an aberrantly modified cell surfaceglycolipid or glycoprotein. It will be appreciated that a tumor antigenmay be expressed by a subset of tumors of a particular type and/or by asubset of cells in a tumor.

Other exemplary therapeutic/diagnostic antibodies that are useful in theproduction of radiolabeled antibodies or proteins according to themethods provided herein include, but are not limited to, the followingantibodies (the target of the antibody is listed in parentheses togetherwith exemplary non-limiting therapeutic indications):

Abciximab (glycoprotein IIb/IIIa; cardiovascular disease), Adalimumab(TNF-α, various auto-immune disorders, e.g., rheumatoid arthritis),Alemtuzumab (CD52; chronic lymphocytic leukemia), Basiliximab (IL-2Rαreceptor (CD25); transplant rejection), Bevacizumab (vascularendothelial growth factor A; various cancers, e.g., colorectal cancer,non-small cell lung cancer, glioblastoma, kidney cancer; wet age-relatedmacular degeneration), Catumaxomab (CD3 and EpCAM, malignant ascites),Cetuximab (EGF receptor, various cancers, e.g., colorectal cancer, headand neck cancer), Certolizumab (e.g., Certolizumab pegol) (TNF alpha;Crohn's disease, rheumatoid arthritis), Daclizumab (IL-2Rα receptor(CD25); transplant rejection), Eculizumab (complement protein C5;paroxysmal nocturnal hemoglobinuria), Efalizumab (CD11a; psoriasis),Gemtuzumab (CD33; acute myelogenous leukemia (e.g., conjugated tocalicheamicin)), Ibritumomab tiuxetan (CD20; Non-Hodgkin lymphoma (e.g.,labeled with yttrium-90 or indium-111)), Infliximab (TNF alpha; variousautoimmune disorders, e.g., rheumatoid arthritis) Muromonab-CD3 (T CellCD3 receptor; transplant rejection), Natalizumab (alpha-4 (α4) integrin;multiple sclerosis, Crohn's disease), Omalizumab (IgE; allergy-relatedasthma), Palivizumab (epitope of RSV F protein; Respiratory SyncytialVirus infection), Panitumumab (EGF receptor; cancer, e.g., colorectalcancer), Ranibizumab (vascular endothelial growth factor A; wetage-related macular degeneration) Rituximab (CD20; non-Hodgkinlymphoma), Tositumomab (CD20; non-Hodgkin lymphoma), Trastuzumab (ErbB2;breast cancer), and any antigen-binding fragments thereof.

In some embodiments, the protein (e.g., antibody) to be radiolabeledbinds a marker of inflammation. In some embodiments the protein to beradiolabeled is a VHH7 or aVHHDC13, which binds murine MHC class IImolecules and CD11b, respectively. The protein (e.g., VHH7 or VHHDC13)may contain additional sequences, including, but not limited to anenzyme recognition sequence (e.g., a sortase recognition sequence), anepitope tag (e.g., a His tag) or a PELB leader sequence. A PELB leadersequence is a sequence of amino acids that, when conjugated to aprotein, directs the protein to the bacterial periplasm, where it isremoved by a signal peptidase. Protein secretion may increase thestability of cloned gene products. In some embodiments the proteins,described herein may contain a PELB comprising the amino acid sequenceMKYLLPTAAAGLLLLAAQPAMA (SEQ ID No: 82). The VHH7 or VHHDC13 protein, maycomprise a sortase recognition motif, a His epitope tag and a PELBleader sequence. In some embodiments, the VHHDC13 is encoded by anucleic acid comprising the nucleic acid sequence set forth in (SEQ IDNO: 83), which encodes the amino acid sequence set forth in (SEQ ID NO:84). In other embodiments, the VHH7 is encoded by a nucleic acidcomprising the nucleic acid sequence set forth in (SEQ ID NO: 85), whichencodes the amino acid sequence set forth in (SEQ ID NO: 86). In otherembodiments, the VHH4 is encoded by a nucleic acid comprising thenucleic acid sequence set forth in (SEQ ID NO: 90), which encodes theamino acid sequence set forth in (SEQ ID NO: 91).

(SEQ ID NO: 83) ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCAGGGGGAGGATTGGTGCAAACTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGTTGACTTCAATTGGTATAGTATGGGGTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATACGTTGCAAGTATAGACCAAGGTGGTGAATTAGATTATGCCATCTCCGTGAAGGGACGATTTACTATCTCCAGAGACAACGCCAAGAACATGGTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATTTCAGCGGGCGCGGCGCGAGTAATCCAGATAAGTATAAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGACTGCCGGAAACCGGCGGCCACCACCATCACCATCACTAATAG. (SEQ ID NO: 84)MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGLVQTGGSLRLSCAASGVDFNWYSMGWFRQAPGKEREYVASIDQGGELDYAISVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAADFSGRGASNPDKYKYWGQGTQVTVSSGGLPE TGGHHHHHH.(SEQ ID NO: 85) ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGCGCAGCCTCTGGACGCACCTTCAGTCGCGGTGTAATGGGCTGGTTCCGCCGGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAATCTTTAGCGGGAGTAGCTGGAGTGGTCGTAGTACATACTATTCAGACTCCGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACGGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCGGGATATCCGGAGGCGTATAGCGCCTATGGTCGGGAGAGTACATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGACTGCCGGAAACCGGCGGCCACCACCATCACCATCACTAATA G. (SEQ ID NO: 86)MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGLVQAGDSLRLSCAASGRTFSRGVMGWFRRAPGKEREFVAIFSGSSWSGRSTYYSDSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYCAAGYPEAYSAYGRESTYDYWGQGTQVTVS SGGLPETGGHHHHHH.(SEQ ID NO: 90) ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCCTCAGTAGCTATGGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAACGTGAAGTGGTCGCAACTATTAGTGCTACTGGTAGCATAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGTGCCAAGAACACGATGTATCTGCAACTGAACAGCCTGACACCTGAGGACACGGCCGTCTATTACTGTAACACAATTTATAGGTCTACTCTCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGACTGCCGGAAACCGGCGGCCACCACCATCACCAT CAC. (SEQ ID NO: 91)MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGLVQAGGSLRLSCAASGSTLSSYGMGWYRQAPGKQREVVATISATGSISYADSVKGRFTISRDSAKNTMYLQLNSLTPEDTAVYYCNTIYRSTLYWGQGTQVTVSSGGLPETGGHHHHH H.

As one example, described in Example 2, radiolabeled VHH7 allowed forthe successful in vivo imaging of immune cells, which localize to lymphtissues, as well as sites of inflammation surrounding tumors. In someembodiments, the radiolabeled proteins (e.g., radiolabeled antibodies orantibody fragments), described herein, may be used to image an immuneresponse. The in vivo imaging of the inflammatory response, e.g., bylabeling sites of inflammation using the methods and compositionsprovided herein, allows for non-invasive diagnosis, monitoring, andtreatment of inflammatory disorders, as described herein. Otherexemplary inflammatory markers to which radiolabeled proteins of theinstant disclosure may bind include, but are not limited to, cytokines,tumor necrosis factor (TNF)-α, IL-6, IL-1 beta, IL-8, IL-10, IL-12,IL-16, IL-18, monocyte chemoattractant protein-1 (MCP-1), GRO-α (GrowthRelated Oncogene-α), matrix metalloproteinase-8 (MMP-8), CSFs(colony-stimulating factors), epithelial cell-derivedneutrophil-activating peptide-78 (ENA-78), regulated on activationnormal T cell expressed and secreted (RANTES) CCL5, CXCL6 (granulocytechemotactic protein-2), CXCL9 MIG, CXCL10; IP-10, CXCL11, CXCL13(BCA-1), Exodus-1 (CCL20), MIF (macrophage migration inhibitory factor):MIP-lalpha (CCL3), MIP-lbeta (CCL4), CD11b, CD11c, CD13, CD15, CD66,CD14, CD64, CD66b, CD18, CD16, CD62L, CD67, HLA-DR, sHLA-G,Dihydroepiandrotendione (DHEA)-S, Cortisol CRF (corticotrophin-releasingfactor), CRF-binding protein, alpha-defensin, beta-defensin, neutrophildefensins (HNP 1-3), bactericidal/permeability-increasing protein (BPI),calprotectin (MRP8/14), surfactant protein-A, surfactant protein-D,serum amyloid P component, serum amyloid A, complement factors,mannan-binding lectin, fibrinogen, prothrombin, factor VIII, vonWillebrand factor, plasminogen, mannan-bindinglectin, c-reactiveprotein, Pentraxin 3, scavenger receptors, C-type lectins, Toll-likereceptor (TLR)-4, TLR-2, TLR-3, TLR-6, intracellular pattern recognitionreceptors (Nod1, Nod2, RIG-1, MDA-5), RAGE (receptor for advancedglycation endproduct), alpha 2-macroglobulin, ferritin, hepcidin,ceruloplasmin, haptoglobin, orosomucoid, alpha 1-antitrypsin, alpha1-antichymotrypsin, lipopolysaccharide-binding protein (LBP), albumin,transferrin (including lactoferrin), transthyretin, retinol-bindingprotein, antithrombin, transcortin, adrenocorticotropin, Urocortin,estriol, MMP-1, MMP-2, TIMP-2, MMP-3, MMP-7, MMP-9, arachidonatelipoxygenase metabolites, prostaglandins, prostacyclins, thromboxanes,leukotrienes, Catalase, Caspase-1 (NALP3 inflammasome), leptin,adiponectin, resistin, visfatin, Retinol binding protein 4 (RBP4),endotoxin, Epidermal growth factor (EGF), insulin-like growth factorbinding protein-1 (IGFBP-1), neutrophil elastase, leukocyte elastase(ELA2, neutrophil), SLPI (secretory leukocyte protease inhibitor), S100calcium binding protein B, Heat shock protein, Endothel in-1, -2,Angiopoietin-2, Calcium-binding protein, Soluble Triggering receptorexpressed on myeloid cells 1 (sTREM1), Protein-Z (vitamin K-dependentplasma glycoprotein), and Tissue factor and Platelet activating factor(PAF).

In other embodiments, the radiolabeled proteins (e.g., radiolabeledantibodies or antibody fragments), described herein, may be used toimage immune cells independent of an immune response. This may be doneusing antibodies that detect specific immune cell markers that are notindicative of an active immune response. As one example, naïve T cellsmay be imaged using any of the radiolabelled antibodies or antibodyfragments, described herein, that bind to the naïve T cell markers CD3,CD4, CD45RA, CD45RB, CD197, or CD62L. Further information on varousimmune cell types may be found in, e.g., Zhu, J., et al.,Differentiation of effector CD4 T cell populations. Annu. Rev. Immunol.,28 (2010), pp. 445-489; S. Crotty, Follicular helper CD4 T cells (TFH),Annu. Rev. Immunol., 29 (2011), pp. 621-663. Of course it would beunderstood that certain of these markers (e.g., CD3, CD4) would also beexpressed on T cells involved in an immune response and could be used astargets for imaging an immune response.

The inventive radiolabeled proteins (e.g., antibodies or antibodyfragments) may be used to non-invasively image tumor and/or T cellmarkers. In some embodiments, the radiolabeled antibodies or antibodyfragments detect markers including, but not limited to PD-L1, PD-1,PD-2, CTLA-4, CD3, CD4, CD8, or CD28.

In certain embodiments, the inventive radiolabeled proteins (e.g.,antibodies or antibody fragments) bind to proteins involved in immunecheckpoint pathways. “Immune checkpoint pathways” or “immunecheckpoints” are naturally existing inhibitory pathways of the immunesystem that play important roles in maintaining self-tolerance andmodulating the duration and level of effector output (e.g., in the caseof T cells, the levels of cytokine production, proliferation or targetkilling potential) of physiological immune responses in order tominimize damage to the tissues of the individual mounting the immuneresponse. Such pathways may, for example, downmodulate T cell activityor enhance regulatory T cell immunosuppressive activity. Examples ofimmune checkpoint pathways include, but are not limited to the PD-1pathway and the CTLA-4 pathway and the TIM3 pathway. Tumors frequentlyco-opt certain immune-checkpoint pathways as a major mechanism of immuneresistance, e.g., against T cells that are specific for tumor antigens.Furthermore, chronic antigen exposure, such as occurs in cancer, canlead to high levels of expression of immune checkpoint proteins (e.g.,PD1, PD-L1, PD-L2) by immune cells, which can induce a state of T cellexhaustion or anergy. Certain immune checkpoint proteins such as CTLA4and PD1 are highly expressed on T regulatory (T_(Reg)) cells and mayenhance their proliferation. Many tumours are highly infiltrated withT_(Reg) cells that likely suppress effector immune responses, Thus,blockade of the PD1 pathway and/or the CTLA4 pathway may enhanceantitumour immune responses by diminishing the number and/or suppressiveactivity of intratumoral T_(Reg) cells. Certain aspects of the inventionutilize the radiolabeled proteins (e.g., radiolabeled antibodies orantibody fragments) for diagnosing or monitoring a disease or condition(e.g., cancer) or the response of a disease or condition (e.g., cancer)to therapy. For example, the radiolebeled antibodies or antibodyfragments may be used to detect whether a tumor expresses an immunecheckpoint marker (e.g., an immune checkpoint protein) and/or to detectwhether a tumor contains immune cells that express an immune checkpointmarker (e.g., an immune checkpoint protein). In other embodiments, theinventive radiolabeled proteins (e.g., radiolabeled antibodies orantibody fragments) bind to an immune checkpoint modulator. In someembodiments the immune checkpoint modulator is an immune checkpointinhibitor. “Immune checkpoint inhibitor” refers to any agent thatinhibits (suppresses, reduces activity of) an immune checkpoint pathway.In some embodiments the immune checkpoint modulator is an immunecheckpoint activator. “Immune checkpoint activator” refers to any agentthat activates (stimulates, increases activity of) an immune checkpointpathway.

Immune checkpoint inhibitors, e.g., monoclonal antibodies that bind toimmune checkpoint proteins such as CTLA4, PD1, PD-L1 have shown notableefficacy in treating a variety of different cancers, including cancersthat are advanced, have failed to respond to conventionalchemotherapeutic agents, and/or have a poor prognosis, such asmetastatic melanoma (see, e.g., Pardoll, D M, The blockade of immunecheckpoints in cancer immunotherapy, Nat Rev Cancer. 2012;12(4):252-64). However, not all subjects with tumors of a particulartype may experience benefit from treatment with a given immunecheckpoint inhibitor. One or ordinary skill would appreciate that abenefit could be, e.g., stable disease rather than progressive disease,eventual reduced number and/or volume of tumor lesions, increased meansurvival, etc. Detection of immune checkpoint markers using any of themethods, described herein, may be used to determine whether or not toadminister a therapeutic and/or to select a therapeutic (e.g., fromamong multiple different therapeutic options). For example, aradiolabeled antibody or antibody fragment that binds PD-L1 can be usedto detect whether a tumor within a patient expresses PD-L1. Patientshaving a PD-L1 positive tumor may then be administered a therapeuticthat targets the PD1 pathway, e.g., a therapeutic (such as an antibody)that targets PD1 or PD-L1. A radiolabeled protein (e.g., radiolabeledantibody or antibody fragment) that binds PD1 can be used to detectwhether a tumor within a patient is positive for PD1 (e.g., due to thepresence of immune cells that express high levels of PD1). Patientshaving a PD1 positive tumor may then be administered a therapeutic agentthat targets the PD1 pathway, In some embodiments a radiolabeled protein(e.g., radiolabeled antibody or antibody fragment) that binds TIM3 canbe used to detect whether a tumor within a patient is positive for TIM3(e.g., due to the presence of immune cells that express high levels ofTIM3). Patients having a TIM3 positive tumor may then be administered atherapeutic that targets the TIM3 pathway, e.g., a therapeutic (such asan antibody) that targets TIM3. In some embodiments a radiolabeledprotein (e.g., radiolabeled antibody or antibody fragment) that bindsCTLA4 can be used to detect whether a tumor within a patient is positivefor CTLA4 (e.g., due to the presence of immune cells that express highlevels of CTLA4). Patients having a CTLA4 positive tumor may then beadministered a therapeutic that targets the CTLA4 pathway, e.g., atherapeutic (such as an antibody) that targets CTLA4. In someembodiments a subject with a tumor may be imaged with two, three, ormore radiolabeled proteins (e.g., antibodies, antibody fragments) thatbind to different immune checkpoint proteins (e.g., proteins involved indifferent immune checkpoint pathways). One or more immune checkpointpathways that are positive in the tumor are identified. The patient isthen treated with one or more agent(s) that target those immunecheckpoint pathways for which a tumor (or one or more tumor(s)) in thesubject is positive. In some embodiments, if the tumor is negative for aparticular immune checkpoint pathway or immune checkpoint protein, analternative treatment may be administered instead of an immunecheckpoint inhibitor that would target that immune checkpoint pathway orimmune checkpoint protein. Other aspects of the invention utilize theradiolabeled antibodies or antibody fragments for monitoring theresponse to a therapeutic or monitoring expression of a protein, such asan immune checkpoint inhibitor protein. For example, a radiolabeledantibody or antibody fragment, described herein, may be used to detectwhether an immune response has been generated or enhanced or suppressedat a site of interest, such as at the site of a tumor or a site ofinfection, or whether the tumor expresses an immune checkpoint protein(e.g., PD-L1). In some embodiments a radiolabeled protein that binds toan immune cell, e.g., a T cell, may be administered to a subject before,concurrently, and/or after administration of a treatment intended toenhance or inhibit an immune response. Images may be compared frombefore and after treatment to assess the effect of the treatment on theimmune response. In some embodiments, the inventive radiolabeledproteins may be used to monitor the response to a therapeutic at leastevery 1 day, at least every 5 days, at least every 10 days, at leastevery 15 days, at least every 30 days, at least every 45 days, at leastevery 60 days, at least every 120 days, at least every 180 days, atleast every 240 days or at least every year. In some embodiments asubject may be monitored for, e.g., up to 3, 6, 9 months, up to 1, 2, 5,years, or more. In some embodiments, a therapeutic agent that targets animmune checkpoint inhibitor pathway is a monoclonal antibody. In someembodiments, the monoclonal antibody is a chimeric, humanized, or humanmonoclonal antibody. In some embodiments, the antibody is an IgGantibody, e.g., an IgG1 or IgG4 antibody. In some embodiments, atherapeutic agent that targets the CTLA4 pathway is a monoclonalantibody that binds to CTLA4, such as ipilimumab (Yervoy) ortremilimumab. In some embodiments, a therapeutic agent that targets thePD1 pathway is a monoclonal antibody that binds to PD1, such asnivolumab (a fully human IgG4 monoclonal antibody), pidilizumab (alsoknown as CT-011, a humanized IgG1 monoclonal antibody), or pembrolizumab(Keytruda, formerly lambrolizumab; also known as MK-3475), a humanizedIgG4 monoclonal antibody), or MEDI0680 (AMP-514, a humanized IgG4mAbagainst PD-1). In some embodiments, a therapeutic agent that targets thePD1 pathway is a monoclonal antibody that binds to PD-L1 such asBMS-936559 (a fully human IgG4 monoclonal antibody), MPDL3280A (humanmonoclonal, Genentech), MSB0010718C (Merck Serono), or MEDI4736. In someembodiments, a therapeutic agent that targets the PD1 pathway is amonoclonal antibody that binds to PD-L2. In some embodiments, atherapeutic agent that targets the PD1 pathway is a recombinant fusionprotein comprising extracellular domain of PD-L2 such as AMP-224. Avariety of PD1 pathway inhibitors, e.g., antibodies that bind to PD-1,PD-L1, or PD-L2 are described in U.S. Pat. Pub. No. 20040213795,20110195068, 20120039906, 20120114649, 20130095098, 20130108651,20130109843, 20130237580, and 20130291136, all of which are incorporatedby reference herein.

In some embodiments, the subject suffers from a solid tumor. In someembodiments, the subject suffers from melanoma, renal cell carcinoma,non-small-cell lung cancer, ovarian cancer, brain cancer (e.g.,glioblastoma), lymphoma (e.g., Non-Hodgkin lymphoma), hepatocellular,esophageal, breast (e.g., triple negative breast cancer), multiplemyeloma, or pancreatic cancer. In some embodiments, the subject has ametastatic cancer, stage III cancer, or stage IV cancer.

It would be understood that the immune checkpoint inhibitor could beadministered as a single agent or in combination with one or more otheranti-tumor agents.

One aspect of the invention relates to radiolabelled proteins (e.g.,antibodies or antibody fragments) that are capable of reaching theirtargets and are cleared quickly from the circulation. Whole antibodiesand their fragments have different characteristics that determine theirtargeting properties, such as how quickly they reach the target antigenand clear from the blood, which organ clears the antibody from theblood, penetration into the tumor and amount of the injectedradiolabeled antibody or antibody fragment binding to the target. Onceantibodies target their respective antigens, they generally bind withhigh avidity, which in turn determines their tumor residence time,whereas the unbound antibody is processed by various organs in the bodyand eventually degraded and excreted. Whole IgG, which is the principalantibody form used, clears very slowly from the blood, requiring severaldays before a sufficient amount leaves the circulation to allow thespecific concentration taken into the tumor to be distinguished fromblood and adjacent tissue radioactivity. Its slow clearance is in partowing to its large size (approximately 150,000 Da) that impedes itsextravasation, resulting in a slow tumor accretion. As the molecularsize of an antibody is reduced from a divalent F(ab′)2 fragment(approximately 100,000 Da) to the monovalent binding Fab fragment(approximately 50,000 Da), there is a progressively faster clearancefrom the blood. Molecular engineering has enabled the formation of evensmaller antibody structures, such as scFv (approximately 25,000 Da),which are cleared even more rapidly from the blood. See Goldenberg D. M.et al., “Novel radiolabeled antibody conjugates.” Oncogene. 2007, 26,3734-3744; the entire contents of which are hereby incorporated byreference. Accordingly, in some embodiments the radiolabeled proteins(e.g., antibodies or antibody fragments), described herein, have amolecular weight of less than 60 kDa, less than 55 kDa, less than 50kDa, less than 45 kDa, less than 40 kDa, less than 35 kDa, less than 30kDa, less than 25 kDa less than 20 kDa, less than 15 kDa, less than 10kDa, or less than 5 kDa. In other embodiments the radiolabelled proteins(e.g., antibodies or antibody fragments), described herein, have amolecular weight ranging from 5 kDa-15 kDa, from 5 kDa-20 kDa, from 5kDa-25 kDa, from 5 kDa-30 kDa, from 5 kDa-35 kDa, from 5 kDa-40 kDa,from 5 kDa-45 kDa, from 5 kDa-55 kDa, from 5 kDa-60 kDa, from 15 kDa-20kDa, from 15 kDa-25 kDa, from 15 kDa-30 kDa, from 15 kDa-35 kDa, from 15kDa-40 kDa, from 15 kDa-45 kDa, from 15 kDa-50 kDa, from 15 kDa-55 kDa,from 15 kDa-60 kDa, from 25 kDa-35 kDa, from 25 kDa-45 kDa, from 25kDa-55 kDa, from 25 kDa-60 kDa, from 35 kDa-45 kDa, from 35 kDa-55 kDa,from 35 kDa-60 kDa, from 45 kDa-55 kDa, from 45 kDa-60 kDa, or from 50kDa-60 kDa. In yet other embodiments the radiolabelled proteins (e.g.,antibodies or antibody fragments), described herein, are expedientlycleared from the circulation following injection into a patient. In someembodiments, at least 95% of the radiolabelled proteins (e.g.,antibodies or antibody fragments) are cleared from the blood within 20minutes, within 30 minutes, within 40 minutes, within 60 minutes, within80 minutes, within 30 minutes, within 40 minutes, within 60 minutes,within 90 minutes, within 2 hours, within 3 hours, within 4 hours,within 6 hours, within 8 hours, within 10 hours or within 12 hours. Inother embodiments, at least 95% of the radiolabelled proteins (e.g.,antibodies or antibody fragments) are cleared from the body within 20minutes, within 30 minutes, within 40 minutes, within 60 minutes, within80 minutes, within 30 minutes, within 40 minutes, within 60 minutes,within 90 minutes, within 2 hours, within 3 hours, within 4 hours,within 6 hours, within 8 hours, within 10 hours or within 12 hours.

In some embodiments, the agent conjugated to a target protein is aprotein, a detectable label, a radiolabeled compound or small molecule,or any other agent described herein. It should be appreciated that theinventive radiolabeled proteins, described herein, may be used fornon-invasive diagnosis of disease, monitoring of disease progression,monitoring of response to treatment, or for the treatment of a disease.

In some embodiments, the methods provided herein further comprisepurifying the labeled protein. Methods for purifying proteins are wellknown in the art, and include those described by Green and Sambrook,Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contentsof which are incorporated herein by reference. In some embodiments, atarget protein may further include a purification tag, detectable label,or both. For example, the fusion protein may comprise a poly-histidinetag (e.g., a 6×His-tag) for purification purposes (e.g., nickel orcobalt affinity based purification; see Hochuli et al., NatureBiotechnology. 1988, 6, 1321-1325). In some embodiments, thepoly-histidine tag comprises 4, 5, 6, 7, 8, 9, or 10 histidines.

According to another embodiment of the invention, methods for modifyinga sortase substrate peptide are provided. Such methods are useful forgenerating sortase substrate peptides having a desired label, such as aradiolabel (e.g., by linking a radioactive agent to the sortasesubstrate peptide). Such modified sortase substrate peptides can then beused to generate radiolabeled proteins using sortagging technology, asdescribed herein. In some embodiments, the method comprises contacting asortase substrate peptide that comprises a nucleophilic group with aradiolabeled agent that comprises an electrophilic group underconditions suitable for the formation of a covalent bond (linkage)between the sortase substrate peptide and agent. In some embodiments,the nucleophilic/electrophilic pairings are any of those suitable foruse in click chemistry, as described herein. In some embodiments, thenucleophilic group is an aminooxy group, a hydrazide, orthiosemicarbazide. In some embodiments, the electrophilic group is acarbonyl group, such as an aldehyde or ketone. As described herein,aminooxy groups, hydrazides, and thiosemicarbazides react with carbonylgroups (e.g., aldehydes and ketones) to form oxime, hydrazone, andthiosemicarbazone linkages, respectively. In some embodiments, the clickchemistry partners are a conjugated diene and an optionally substitutedalkene, In other embodiments, the click chemistry partners are anoptionally substituted tetrazine and an optionally substitutedtrans-cyclooctene (TCO). In some embodiments, the click chemistryreactive pair includes tetrazine (Tz) and trans-cyclooctene (TCO). Inother embodiments, the click chemistry partners are an alkyne and anazide. For example, a difluorinated cyclooctyne, a dibenzocyclooctyne, abiarylazacyclooctynone, or a cyclopropyl-fused bicyclononyne can bepaired with an azide as a click chemistry pair. In other embodiments,the click chemistry partners are reactive dienes and suitable tetrazinedienophiles. For example, TCO, norbornene or biscyclononene can bepaired with a suitable tetrazine dienophile as a click chemistry pair.In yet other embodiments, tetrazoles can act as latent sources ofnitrile imines, which can pair with unactivated alkenes in the presenceof ultraviolet light to create a click chemistry pair, termed a“photo-click” chemistry pair. Other suitable click chemistry handles areknown to those of skill in the art (See e.g., Table 1; Spicer et al.,“Selective chemical protein modification.” Nature Communications. 2014;5:4740). For two molecules to be conjugated via click chemistry, theclick chemistry handles of the molecules have to be reactive with eachother, for example, in that the reactive moiety of one of the clickchemistry handles can react with the reactive moiety of the second clickchemistry handle to form a covalent bond. Such reactive pairs of clickchemistry handles are well known to those of skill in the art andinclude, but are not limited to, those described in Table 1.

In some embodiments, the agent linked to the sortase substrate is anyradioactive molecule or compound useful for diagnostic and/ortherapeutic applications, such as PET or SPECT, that can be conjugatedto a sortase substrate peptide using nucleophile/electrophile pairs. Forexample, in some embodiments, a sortase substrate peptide and an agentto be conjugated each comprise click chemistry handles (e.g., asdescribed herein) capable of specifically interacting with each other toform a covalent bond. In other embodiments, the sortase substratepeptide comprises an aminooxy group, a hydrazide, or thiosemicarbazide.For example, in some embodiments, a sortase substrate peptide issynthesized to an include a C-terminal amino acid comprising an aminooxygroup, a hydrazide, or thiosemicarbazide (e.g., lysine- (K-)aminooxy;see, e.g., Examples). Such a sortase substrate peptide reacts with aradioactive agent comprising a carbonyl group. In some embodiments, theradioactive agent comprising a carbonyl group (e.g., aldehyde, ketone,etc.) is a carbohydrate comprising any radionuclide (isotope) useful indiagnostic and/or therapeutic applications, for example those describedherein. In some embodiments, the radioactive agent is FDG or¹⁴C—(U)-glucose. As described herein, FDG and ¹⁴C—(U)-glucose (and otherreducing sugars) isomerize in solution to form an open-chain moleculehaving an aldehyde that can react with a nucleophilic group of a sortasesubstrate peptide (e.g., as described herein). In some embodiments, thesortase peptide substrate comprises a tetrazine handle and is reactedwith an agent comprising a trans-cyclooctene handle. In otherembodiments, the sortase peptide substrate comprises an azide handle andis reacted with an agent comprising a difluorinated cyclooctyne, adibenzocyclooctyne, a biarylazacyclooctynone, or a cyclopropyl-fusedbicyclononyne handle. In certain embodiments, a sortase substratecomprises a tetrazine dienophile handle and is reacted with an agentcomprising a TCO, a norbornene or a biscyclononene handle. In yet otherembodiments, a sortase substrate comprises a nitrile imine handle and isreacted with an agent comprising an unactivated alkenes in the presenceof ultraviolet light. In certain embodiments, a sortase substratecomprises a cysteine handle and is reacted with an agent comprising amaleimide. For example the cysteine from a peptide (e.g., GGGC) may bereacted with a maleimide that is associated with a chelating agent(e.g., NOTA).

In some embodiments, the sortase substrate peptide comprises a chelator.As one example, a sortase substrate peptide (e.g., GGGC) may be reactedwith maleimide-NOTA to generate the sortase substrate peptide fused to achelator (e.g., GGG-NOTA). The sortase substrate peptide, comprising achelator, can be tethered to a protein of interest, having a sortaserecognition motif (e.g., LPXTG) using a sortase-mediatedtranspeptidation reaction. The protein of interst fused to the chelatorcan then be treated with a radiolabel that binds the chelator (e.g.,⁶⁴Cu) to generate a radiolabeled protein. For example, the sortasesubstrate peptide GGG-NOTA may be fused to a VHH protein having a LPXTGsortase recognition motif to generate a VHH protein that is fused to theNOTA chelator (e.g., VHH-NOTA). The VHH-NOTA molecule can then betreated with ⁶⁴Cu to generate a ⁶⁴Cu labeled VHH protein. In otherembodiments, the sortase substrate peptide, comprising a chelator, isfirst treated with a radiolabel (e.g., ⁶⁴Cu) to generate a radiolabeledsubstrate peptide, which may then be tethered to a protein of interest,having a sortase recognition motif (e.g., LPXTG) using asortase-mediated transpeptidation reaction. It should be appreciatedthat the chelator may be any chelating molecule known in the art and theexamples provided are not meant to be limiting. Accordingly, the metalbound by the chelator may be any suitable metal or radiolabeled metalknown in the art capable of binding to the chelator.

In some embodiments, the sortase substrate peptide comprises anN-terminal sortase recognition motif, e.g., as described herein. Inother embodiments, the sortase substrate peptide comprises a C-terminalsortase recognition motif, e.g., as described herein. In someembodiments, the sortase substrate peptide (e.g., having either anN-terminal or C-terminal sortase recognition motif) further comprises anucleophilic group, e.g., as described herein. In some embodiments, thesortase substrate peptide comprises an oligoglycine or an oligoalaninesequence, for example 1-10 N-terminal glycine residues or 1-10N-terminal alanine residues, respectively. In some embodiments, thesortase substrate peptide comprises the sequence GGG. In someembodiments, the sortase substrate peptide comprises the sequence(G)_(n1)K, wherein n1 is an integer between 1 and 10, inclusive. In someembodiments, the sortase substrate peptide comprises the sequence GGGK(SEQ ID NO:81), wherein the lysine (K) is modified to include anucleophilic group, e.g., as provided herein. In some embodiments, thelysine (K) is modified to include a click chemistry handle. In someembodiments, the lysine (K) is modified to include an aminoozy group, ahydrazide, or thiosemicarbazide. In some embodiments, the sortasesubstrate peptide comprises the sequence GGGK (SEQ ID NO:81), whereinthe lysine (K) is modified to include an aminooxy group. In someembodiments, the sortase substrate peptide comprises an N-terminalsortase recognition motif, wherein any amino acid or other constituentof the substrate comprises a nucleophilic group as described herein. Forexample, in some embodiments, the sortase substrate peptide comprisesthe sequence (G)_(n1)X, wherein n1 is an integer between 1 and 10,inclusive, and X is any amino acid, click chemistry handle, molecule, orcompound having a nucleophilic group, e.g., as described herein. Methodsfor generating sortase substrate peptides comprising modified aminoacids (e.g., modified to include nucleophilic groups) are known, andinclude peptide coupling reactions (e.g., synthesis). See, e.g., Nilssonet al., “Chemical Synthesis of Proteins.” Annu. Rev. Biophys. Biomol.Struct. 2005; 34: 91-118; the entire contents of which are herebyincorporated by reference.

In some embodiments, the methods for modifying a sortase substratepeptide further involve the use of one or more catalysts. For example,because of the need to quickly and efficiently generate radioactivesortase substrate peptides, in some embodiments the use of a catalystresults in a fast and/or more efficient coupling reaction between thesortase substrate peptide and (radioactive) agent. Typically, themethods involve contacting the sortase substrate peptide and/or agentwith a catalyst. In some embodiments, the catalyst is any catalystcapable of decreasing the reaction time and/or increasing the efficiencyof the coupling reactions (e.g., the nucleophilic/electrophilicpairings) between sortase substrate peptides and (radioactive) agentsdescribed herein. The catalyst will be chosen based on the functionalgroups and chemistry being used to couple the sortase substrate peptideto the agent. For example, with use of certain click chemistry reactionssuch as azide-alkyne (e.g., Huisgen) cycloaddition (see Table 2), one ofskill would choose a copper based catalyst. In some embodiments, the oneor more catalysts is (are) chosen from the non-limiting list thatincludes m-phenylenediamine (mPDA), o-phenylenediamine,p-phenylenediamine, o-aminophenol, m-aminophenol, p-aminophenol,o-aminobenzoic acid, 5-methoxyanthranilic acid, 3,5-diaminobenzoic acidor aniline. In some embodiments, the catalyst is m-phenylenediamine(mPDA).

As described herein, it can be advantageous to modify a sortasesubstrate peptide in as short a time frame as possible, especially whenconjugating (linking) a radioactive agent to the substrate that has arelatively short half-life. Thus, in some embodiments, the methodsprovided herein allow for the sortase substrate peptide to be modified(e.g., linked to a radioactive agent) in less 5 minutes, less than 10minutes, less than 15 minutes, less than 20 minutes, less than 25minutes, less than 30 minutes, less than 45 minutes, less than 60minutes, less than 90 minutes, or less than 120 minutes. In someembodiments, the sortase substrate peptide is modified in about 1-30minutes, about 2-25 minutes, about 3-20 minutes, about 4-15 minutes, orin about 5-10 minutes. In some embodiments, the methods provided hereinallow for at least 90%, at least 95%, or at least 98% of the sortasesubstrate peptide to be covalently linked to an agent.

Methods of Using Sortagged, Radiolabeled Proteins

In another aspect, provided are methods of obtaining a radiologic imageof a specific tissue or organ of a subject, comprising: (i)administering the radioactive protein as described herein, or thepharmaceutical composition thereof, to the subject; (ii) obtaining theradiologic image of the tissue or organ by capturing the radiationemitted. In certain embodiments, the gamma radiation is emitted. Theprovided imaging methods can facilitate diagnosing, monitoring, ortreating a subject in need thereof. In certain embodiments, theradioactive protein or composition thereof is prepared and administeredshortly before the imaging collection step.

The subject is typically a mammalian subject, e.g., a human. In someembodiments, the subject is a non-human animal that serves as a modelfor a disease or disorder that affects humans. The animal model may beused, e.g., in preclinical studies, to assess efficacy and/or determinea suitable dose.

In some aspects, the instant disclosure relates to the increasingawareness of the interplay between host stromal cells, tumor cells, andmigratory cells, such as macrophages and lymphocytes, and raises anumber of possibilities for non-invasive diagnosis of diseases anddisorders, including cancers and other proliferative diseases as well asinflammatory disorders (Forssell et al., Clin. Cancer Res. 2007, 13,1472-1479; Allavena et al., Crit. Rev. Oncol. Hematol. 2008, 66, 1-9).For example, activated macrophages are often present at the tumormargin. Depending on their functional properties (M1 or M2-type),macrophages help establish a microenvironment either detrimental orfavorable to tumor growth. Therefore, the ability to non-invasivelyimage their presence, as described in Example 2, using anti-Class II MHCantibody fragments, represents a significant improvement over prior artmethods for the diagnosis, monitoring, and treatment of disease. Currentmethods rely on sampling peripheral blood to enumerate and characterizecells in the circulation, or more invasive strategies such ashistological analysis of biopsies or at necropsy. However, as noted byother investigators: “Invasive measurements are prone to sampling errorsand are poorly reflective of the dynamic changes in the location,number, and movement of lymphoid cells. These limitations indicate theneed for non-invasive whole-body imaging methodologies that allowlongitudinal, quantitative, and functional analyses of the immune systemin vivo” (Nair-Gill et al., Immunol. Rev. 2008, 221, 214-228). Sites ofan ongoing inflammatory or immune response provide clues as to theexistence and location of tumor foci, which a biopsy can then confirm.

Prior art methods for protein (e.g., antibody) labeling have revolvedmostly around chemical modification with metal chelators to enableinstallation of radioisotopes such as ⁶⁴Cu, ⁶⁸Ga, or ⁸⁹Zr (Tanaka etal., Org. Biomol. Chem. 2008, 6, 815; Fani et al., Contrast Media Mol.Imaging 2008, 3, 53-63; Vosjan et al., Nat. Protoc. 2010, 5, 739-743).However, the creation of fluorine-carbon bonds in a manner that allowsfacile ¹⁸F labeling without causing collateral damage to proteins hasremained a synthetic challenge (Furuya et al., Curr. Opin. Drug Discov.Devel. 2008, 11, 803-819; Truong et al., J. Am. Chem. Soc. 2013, 135,9342-9345). ¹⁸F PET, relative to other available PET isotopes such ⁶⁸Ga,⁶⁴Cu, ⁸⁹Zr, and ¹²⁴I, has four major advantages: lower energy positronemission, shorter half-life, reduced cost, and wider availability. While¹⁸F PET imaging is used for diagnostic purposes and to monitortherapeutic efficacy, these approaches have relied mostly on agents thatreport on metabolic activity, such as ¹⁸F-fluorodeoxyglucose (FDG) orlabeled precursors in the biosynthesis of nucleic acids, to scorereductions in tumor size (Bohnen et al., J. Nucl. Med. 2012, 53, 59-71;Groheux et al., Eur. J. Nucl. Med. Mol. Imaging 2011, 38, 426-435;Youssef et al., J. Nucl. Med. 2012, 53, 241-248; Waldherr et al., J.Nucl. Med. Off. Publ. Soc. Nucl. Med. 2005, 46, 114-120).

While the methods provided herein are amenable for labeling any protein(e.g., any type of antibody or antibody fragment), the use of singledomain VHHs offers a number of advantages: they are smaller in size (˜15kDa) than Fab (˜50 kDa), ScFv (˜25 kDa), and “diabody” (a pair of scFvsconnected to a pair of C_(H)3 domains; ˜60 kDa) antibody derivatives,and VHHs lack an Fc portion. Technology to humanize these camelid VHHshas been developed (see e.g., Vincke et al., J. Biol. Chem. 2009, 284,3273-3284; which is incorporated herein by reference), and several VHHshave been used already in a number of phase I and phase II clinicaltrials for therapeutic applications (see, e.g., De Meyer et al., TrendsBiotechnol. 2014, 32, 263-270; which is incorporated herein byreference). Accordingly, in some embodiments, methods for usingradiolabeled VHHs (e.g., those labeled according to the methods providedherein) are provided.

In some embodiments, the methods comprise (a) administering any of thecompositions comprising a radiolabeled protein described herein (e.g.,those comprising pharmaceutically acceptable carriers) to a subject; and(b) detecting the radiolabel in the subject. The step of detecting maybe carried out by any procedure known in the art that may allow theimaging of the radiolabeled protein that is administered. For example,in some embodiments, detecting (e.g., imaging) may be carried out by PETand/or single-photon emission computed tomography (SPECT) imaging. Inother embodiments, the imaging may be carried out by both PET and SPECTor by combined imaging methods such as PET/CT (PET with concurrentcomputed tomography imaging) or PET/MRI (PET with concurrent magneticresonance imaging). The imaging procedure may result in one or moreimages of the region of observation of the subject, and in embodimentsin which imaging results in more than one image, these multiple imagesmay be combined, overlaid, added, subtracted, color coded or otherwisefused and mathematically manipulated by any method known in the art. Theimage produced may be a digital or analog image that may be displayed asa “hard” image on, for example, printer paper, photographic paper orfilm, or as an image on a screen, such as for example, a video or LCDscreen.

The images produced using the imaging procedure embodied in the presentinvention may be analyzed by any method known in the art. For example,in one embodiment, the image may be reviewed by a physician or anothermedical professional who visually observes the derived images and gradesthe disease state based on the observable presence of radiolabeledprotein in the images produced. In another embodiment, the images may beanalyzed by a processor or processor system. For example, in oneembodiment, image data derived from a PET or SPECT scan can be inputtedinto a processor that identifies individual pixels or groups of pixelswhose brightness is greater than a predetermined threshold or an averagebackground, and identified pixels may be characterized as indicating thepresence of a radiopharmaceutical. In another embodiment, the image datamay be derived from images scanned and inputted into a processor. Insuch embodiments, a similar process that identifies bright spots on theimage may be used to locate the radiopharmaceuticals in the image. Incertain embodiments, the analysis of the image may further includedetermining the intensity, concentration, strength or combinationthereof of the output brightness, which may be correlated to the amountof radiolabeled protein in the image, an area or region of the image, ora particular spot on the image. Without wishing to be bound by theory,an area or spot on an image having a greater intensity than other areasor spots may hold a higher concentration of radiolabeled proteintargeted to, for example, a tumor, and thus may have a higherconcentration of the radioisotope attached to the region where theradiolabeled protein localizes. Images may also be analyzed by thespatial location of regions of interest to which the administeredradiolabeled proteins are targeted. In other embodiments, analysis ofthe pharmacokinetics of the administered radiolabeled proteins mayprovide information on the appropriate timing of injection of theradiolabeled protein. By identifying areas, regions, or spots on animage that correlate to the presence of a radiolabeled protein, thepresence or absence of a diseased state may be determined. For example,in embodiments in which the protein binds a tumor cell, atumor-associated cell (e.g., neovasculature cell), or a tumor antigen,identifying regions or spots where such protein concentrates indicatesthe presence of a tumor. In some embodiments, images that correlate tothe presence of a radiolabeled protein are used to assess the state ofan inflammatory response, e.g., in a disease or disorder involvinginflammation. Accordingly, the methods provided herein are amenable fordiagnosing, prognosing, or otherwise monitoring an inflammatoryresponse. Exemplary inflammatory diseases/disorders include, but are notlimited to, inflammation associated with acne, anemia (e.g., aplasticanemia, haemolytic autoimmune anaemia), asthma, arteritis (e.g.,polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu'sarteritis), arthritis (e.g., crystalline arthritis, osteoarthritis,psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoidarthritis and Reiter's arthritis), ankylosing spondylitis, amylosis,amyotrophic lateral sclerosis, autoimmune diseases, allergies orallergic reactions, atherosclerosis, bronchitis, bursitis, cancer,chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructivepulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., typeI diabetes mellitus, type 2 diabetes mellitus), a skin condition (e.g.,psoriasis, eczema, burns, dermatitis, pruritus (itch)), endometriosis,Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasakidisease, glomerulonephritis, gingivitis, hypersensitivity, headaches(e.g., migraine headaches, tension headaches), ileus (e.g.,postoperative ileus and ileus during sepsis), idiopathicthrombocytopenic purpura, interstitial cystitis (painful bladdersyndrome), gastrointestinal disorder (e.g., selected from peptic ulcers,regional enteritis, diverticulitis, gastrointestinal bleeding,eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis,eosinophilic gastritis, eosinophilic gastroenteritis, eosinophiliccolitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, orits synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn'sdisease, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminatecolitis) and inflammatory bowel syndrome (IBS)), lupus, multiplesclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephroticsyndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers,polymyositis, primary biliary cirrhosis, neuroinflammation associatedwith brain disorders (e.g., Parkinson's disease, Huntington's disease,and Alzheimer's disease), prostatitis, chronic inflammation associatedwith cranial radiation injury, pelvic inflammatory disease, reperfusioninjury, regional enteritis, rheumatic fever, systemic lupuserythematosus, schleroderma, scierodoma, sarcoidosis,spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantationrejection, tendonitis, trauma or injury (e.g., frostbite, chemicalirritants, toxins, scarring, burns, physical injury), vasculitis,vitiligo and Wegener's granulomatosis.

Compositions

According to other aspects, sortase-mediated radiolabeled proteins,radiolabeled sortase substrate peptides, and compositions thereof areprovided. In some embodiments, compositions for labeling proteins and/orsortase substrate peptides are provided. In some embodiments, providedherein is a pharmaceutical composition comprising the radioactiveprotein as described herein, or a pharmaceutically acceptable salt,solvate, hydrate, tautomer, stereoisomer, isotopically labeledderivative, or prodrug thereof, as described herein, and apharmaceutically acceptable carrier.

For example, in some embodiments, the radiolabeled proteins generatedaccording to any of the inventive methods described herein, are providedin an effective amount in the pharmaceutical composition. In certainembodiments, the effective amount is a diagnostically effective amount.In certain embodiments, the effective amount is sufficient for a medicalprofessional to obtain one or more images of an organ or tissue in asubject.

In some embodiments, the protein is linked (conjugated) to a radioactiveagent, e.g., by means of the inventive methods provided herein involvingsortagging technology. In some embodiments, the agent is a carbohydrate,e.g., a sugar. In some embodiments, the agent is FDG or ¹⁴C—(U)-glucose.In some embodiments, the agent comprises one or more radionuclide(s)suitable for use in diagnostic and/or therapeutic applications (e.g.,PET). In some embodiments, the one or more radionuclides is (are)carbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82,copper-61, copper-62, copper-64, yttrium-86, gallium-68, zirconium-89,or iodine-124. In some embodiments, the agent is labeled with aradionuclide that is fluorine-18.

In some embodiments, the protein is any protein having or engineered tohave a sortase recognition motif. In some embodiments, the protein isany diagnostic or therapeutic protein including, but not limited to,those described herein (e.g., an antibody, an affibody, a single-domainantibody, a Fab fragment, or a therapeutic peptide). In someembodiments, the protein comprises a VHH domain (e.g., VHH4, VHH7). Insome embodiments, the protein is one that binds to a tumor cell, atumor-associated cell (e.g., neovasculature cells) or a tumor antigen.In some embodiments, the protein is one that binds a marker ofinflammation. In some embodiments, the protein is any antibody describedherein, including those disclosed in Table 4 of Salsano and Treglia,“PET imaging using radiolabeled antibodies: future direction in tumordiagnosis and correlate applications.” Research and Reports in NuclearMedicine. 2013: 3; 9-17, and/or those described herein and disclosed inWright and Lapi, “Designing the magic bullet? The advancement ofimmuno-PET into clinical use.” J. Nucl. Med. 2013 August; 54(8):1171-4.

In some embodiments, the composition comprises an amount ofradioactivity suitable for use in diagnostic and/or therapeuticapplications, for example PET. In some embodiments, the compositioncomprises at least 10, at least 20, at lease 30, at least 40, at least50, at least 75, at least 100, at least 150, at least 200, at least 250,at least 300, at least 350, at least 400, at least 450, at least 500, atleast 550, at least 600, at least 700, at least 800, at least 900, or atleast 1000 MBq of radioactivity.

In some embodiments, provided compositions further comprise apharmaceutically acceptable carrier. Thus, in some embodiments, theinvention provides pharmaceutical compositions comprising any of thelabeled proteins described herein, for example, a protein that has beenconjugated to a radiolabeled agent via sortagging technology.

A pharmaceutical composition may comprise a variety of pharmaceuticallyacceptable carriers. Pharmaceutically acceptable carriers are well knownin the art and include, for example, aqueous solutions such as water, 5%dextrose, or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil, orinjectable organic esters that are suitable for administration to ahuman or non-human subject. See, e.g., Remington: The Science andPractice of Pharmacy, 21^(st) edition; Lippincott Williams & Wilkins,2005. In some embodiments, a pharmaceutically acceptable carrier orcomposition is sterile. A pharmaceutical composition can comprise, inaddition to the active agent (e.g., radiolabeled protein),physiologically acceptable compounds that act, for example, as bulkingagents, fillers, solubilizers, stabilizers, osmotic agents, uptakeenhancers, etc. Physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose, lactose; dextrans;polyols such as mannitol; antioxidants, such as ascorbic acid orglutathione; preservatives; chelating agents; buffers; or otherstabilizers or excipients. The choice of a pharmaceutically acceptablecarrier(s) and/or physiologically acceptable compound(s) can depend forexample, on the nature of the active agent, e.g., solubility,compatibility (meaning that the substances can be present together inthe composition without interacting in a manner that would substantiallyreduce the pharmaceutical efficacy of the pharmaceutical compositionunder ordinary use situations) and/or route of administration of thecomposition. The pharmaceutical composition could be in the form of aliquid, gel, lotion, tablet, capsule, ointment, cream, transdermalpatch, etc. A pharmaceutical composition can be administered to asubject by various routes including, for example, parenteraladministration. Exemplary routes of administration include intravenousadministration; respiratory administration (e.g., by inhalation),intramuscular administration, nasal administration, intraperitonealadministration, oral administration, subcutaneous administration andtopical administration. For oral administration, the agent(s) can beformulated with pharmaceutically acceptable carriers as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Insome embodiments an active agent may be administered directly to atarget tissue. Direct administration could be accomplished, e.g., byinjection or by implanting a sustained release implant within thetissue. Of course a sustained release implant could be implanted at anysuitable site. In some embodiments, a sustained release implant may beparticularly suitable for prophylactic treatment of subjects at risk ofdeveloping a recurrent cancer. In some embodiments, a sustained releaseimplant delivers therapeutic levels of the active agent for at least 30days, e.g., at least 60 days, e.g., up to 3 months, 6 months, or more.One skilled in the art would select an effective dose and administrationregimen taking into consideration factors such as the patient's weightand general health, the particular condition being treated, etc.Exemplary doses may be selected using in vitro studies, tested in animalmodels, and/or in human clinical trials as standard in the art.

A pharmaceutical composition comprising a radioactive protein accordingto aspects of this invention may be delivered in an effective amount, bywhich is meant an amount sufficient to achieve a biological response ofinterest, e.g., reducing one or more symptoms or manifestations of adisease or condition. In some embodiments, an effective amount is theamount required to visualize, detect, or identify a given tissue usingdiagnostic procedures such as PET. The exact amount required will varyfrom subject to subject, depending on factors such as the species, age,weight, sex, and general condition of the subject, the severity of thedisease or disorder, the particular labeled protein and its activity,its mode of administration, concurrent therapies, and the like. In someembodiments, a compound, e.g., a protein, is formulated in unit dosageunit form for ease of administration and uniformity of dosage, whichterm as used herein refers to a physically discrete unit of agentappropriate for the patient to be treated. It will be understood,however, that the total daily dosage will be decided by the attendingphysician within the scope of sound medical judgment.

According to some embodiments, sortase substrate peptides linked to aradiolabeled agent, and compositions comprising such, are provided. Insome embodiments, sortase substrate peptides linked to a reactivemoiety, such as a click chemistry handle (e.g., tetrazine), areprovided. In some embodiments, the sortase substrate peptide is anysortase substrate peptide generated according to the inventive methodsprovided herein. In some embodiments, the sortase substrate peptide andagent are linked by an oxime, a hydrazone, a thiosemicarbazone, aheterocyclylene linkage, an amide linkage, an ester linkage, an etherlinkage, a disulfide linkage or through use of click chemistry e.g., asdescribed herein. In some embodiments, the agent is a carbohydrate,e.g., a sugar. In some embodiments, the agent is FDG or ¹⁴C—(U)-glucose.In some embodiments, the agent comprises one or more radionuclide(s)suitable for use in diagnostic and/or therapeutic applications (e.g.,PET). In some embodiments, the one or more radionuclides is (are)carbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82,copper-61, copper-62, copper-64, yttrium-86, gallium-68, zirconium-89,or iodine-124. In some embodiments, the agent is labeled with aradionuclide that is fluorine-18.

In some embodiments, the sortase substrate peptide comprises either anN-terminal or C-terminal sortase recognition motif, e.g., any of thosedescribed herein. In some embodiments, the sortase substrate peptidecomprises an oligoglycine or an oligoalanine sequence, for example 1-10N-terminal glycine residues or 1-10 N-terminal alanine residues,respectively. In some embodiments, the sortase substrate peptidecomprises the sequence GGG. In some embodiments, the sortase substratepeptide comprises the sequence (G)_(n1)K, wherein n1 is an integerbetween 1 and 10, inclusive. In some embodiments, the sortase substratepeptide comprises the sequence GGGK (SEQ ID NO:81). In some embodiments,the sortase substrate peptide and agent are joined by an oxime linkage.In some embodiments, the sortase peptide substrate is linked to an agentthat is or comprises FDG or ¹⁴C—(U)-glucose.

According to some embodiments, compositions for use in the inventivemethods described herein are provided. For example, in some embodiments,compositions for use in radiolabeling a protein using sortaggingtechnology are provided. Typically, the compositions comprise aradiolabeled sortase substrate peptide (e.g., as described herein), asortase (e.g., as described herein), and a protein to be labeled (e.g.,as described herein) comprising a sortase recognition motif. In someembodiments, the protein comprises a C-terminal sortase recognitionmotif (e.g., as described herein), and the sortase substrate peptidecomprises an N-terminal sortase recognition motif (e.g., as describedherein). In some embodiments, the protein comprises an N-terminalsortase recognition motif, and the sortase substrate peptide comprises aC-terminal sortase recognition motif. In some embodiments, the sortasesubstrate is linked (conjugated) to any radioactive agent describedherein (e.g., FDG, ¹⁴C—(U)-glucose, ¹⁸F, etc.). In some embodiments, thesortase is any sortase described herein (e.g., sortase A fromStaphylococcus aureus (SrtA_(aureus)), sortase A from Streptococcuspyogenes (SrtA_(pyogenes)), sortase B from S. aureus (SrtB_(aureus)),sortase B from Bacillus anthracis (SrtB_(anthracis)), or sortase B fromListeria monocytogenes (SrtB_(monocytogenes)). In some embodiments, theprotein is any protein comprising (or engineered to comprise) a sortaserecognition motif (e.g., an antibody, an affibody, a single-domainantibody, a Fab fragment, or a therapeutic peptide).

In some embodiments, compositions for use in modifying sortase substratepeptides are provided. In some embodiments, the composition comprisessortase substrate peptide (e.g., comprising a nucleophilic group asdescribed herein) and a radioactive agent (e.g., comprising anelectrophilic group as described herein). In some embodiments, thecomposition further comprises a catalyst (e.g., m-phenylenediamine(mPDA), o-phenylenediamine, p-phenylenediamine, o-aminophenol,m-aminophenol, p-aminophenol, o-aminobenzoic acid, or aniline).

In certain embodiments, the pharmaceutical compositions provided asdescribed herein are of liquid dosage forms for oral or parenteraladministration. In certain embodiments, the pharmaceutical compositionsare pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as Cremophor™,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension, or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, (e) solutionretarding agents such as paraffin, (f) absorption accelerators such asquaternary ammonium compounds, (g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolinand bentonite clay, and (i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may include a buffering agent.

Kits

Some aspects of this invention provide kits useful for labeling proteinswith radioactive agents using sortagging technology, and/or forgenerating radiolabeled sortase substrate peptides.

In some embodiments, the kit comprises a sortase substrate peptidecomprising a nucleophilic group (e.g., those described herein), amodifying agent that comprises an electrophilic group (e.g., thosedescribed herein), and a catalyst. In some embodiments, the nucleophilicgroup is an aminooxy group, a hydrazide, a thiosemicarbazide, or a clickchemistry handle. In some embodiments, the electrophilic group is analdehyde, a ketone, or a click chemistry handle. In some embodiments,the electrophilic/nucleophilic pairings are tetrazine (Tz) andtrans-cyclooctene (TCO). In some embodiments, the sortase substratepeptide comprises the sequence GGGK, wherein the K is modified toinclude a nucleophilic group (e.g., an aminooxy group, a hydrazide, athiosemicarbazide, or a click chemistry handle). In some embodiments,the modifying agent is any radioactive agent described herein (e.g.,FDG, ¹⁴C—(U)-glucose, ¹⁸F—NaF, etc.). In some embodiments, the catalystis m-phenylenediamine (mPDA), o-phenylenediamine, p-phenylenediamine,o-aminophenol, m-aminophenol, p-aminophenol, o-aminobenzoic acid,5-methoxyanthranilic acid, 3,5-diaminobenzoic acid or aniline.

In some embodiments, a provided kit comprises a sortase substratepeptide that comprises or is linked to a radioactive agent (e.g., any ofthose described herein). In some embodiments, the kit comprises asortase substrate peptide linked to an agent via an oxime linkage, and asortase.

In some embodiments, the kit comprises one or more sortase(s) providedherein. In some embodiments, the sortase is sortase A fromStaphylococcus aureus (SrtA_(aureus)), sortase A from Streptococcuspyogenes (SrtA_(pyogenes)), sortase B from S. aureus (SrtB_(aureus)),sortase B from Bacillus anthracis (SrtB_(anthracis)), or sortase B fromListeria monocytogenes (SrtB_(monocytogenes)).

In some embodiments, the kit comprises a radiolabeled protein generatedaccording to an inventive method provided herein.

In some embodiments, the kit further comprises a buffer or reagentuseful for carrying out a sortase-mediated transpeptidation reaction,for example, a buffer or reagent described in the Examples section.

The following working examples are intended to describe exemplaryreductions to practice of the methods, reagents, and compositionsprovided herein and do not limited the scope of the invention.

EXAMPLES Example 1. Site-Specific Labeling of Proteins Using Glucose,2-Deoxy-2-Fluoroglucose (FDG), and U-¹⁴C-Glucose

In a first step, a short synthetic peptide (Gly)₃-R (1, FIG. 1B) wasgenerated, where R contains an aminooxy functionality that enables anoxime ligation reaction with a ¹⁸F-FDG; this compound occurs both in ahemiacetal cyclical form and as a linear aldehyde. The dynamicequilibrium between these two forms allowed the installation of¹⁸F-glucose on the (Gly)₃-R peptide. The reaction was optimized usingnon-isotopically labeled glucose, and reaction products werecharacterized by LC-MS (FIGS. 1B, 1C, & 1D). In this approach, theaminooxy-functionalized peptide was incubated with glucose (or otheraldehydes) in the presence of a catalyst at pH 5 with constant agitationat 100° C. for 5 to 10 min. The actual timing was determined by theconcentrations of the catalyst, aldehyde and aminooxy compounds used ineach specific reaction. The reaction was cooled on ice for 30 s andquickly added to a pre-prepared solution containing the protein ofinterest and sortase at 30° C. After 3 min of incubation, Ni-NTA beadswere added and the reaction mixture was agitated for another 3 min at30° C. The protein being labeled contained a polyhistidine tagC-terminal of the Gly in the LPXTG (SEQ ID NO:1) motif, which was lostfrom the desired product upon successful transacylation. The sortaseitself also contained a His6 tag, so that sortase and any remainingunreacted proteins could be removed by adsorption to Ni-NTA resin, whilethe labeled protein remained free in solution. Ni-NTA beads were removedby centrifugation; the supernatant was recovered and loaded on asize-exclusion desalting spin column, pre-washed with phosphate buffer.Centrifugation at 1000×g for 30 s provided the final purifiedlabeled-protein of interest. In view of the short (110 min) half-life of¹⁸F, it was important to keep the labeling strategy as short as possibleto obtain maximum radiochemical yields without compromising purity andease of preparation. Using this methodology, >95% labeling was achievedwhen incubating 1 mM of the Gly₃-aminooxy probe with 40 mM of glucose at100° C. for 5 min. m-phenylenediamine (mPDA) was used as a catalyst,which is one of the most efficient of a series of catalysts that cancatalyze the oxime ligation. Due to the fact that the pKa of mPDA is4.9, the pH of the reaction was set between 5-5.3 to maximize thereaction rate.

After establishing the above method using glucose, the aminooxy probewas modified with 2-deoxy-2-fluoroglucose (FDG). Similarly, >90% yieldof FDG-labeled proteins was achieved in as little as 15 min followingthe methodology summarized above (FIG. 2). Product formation wasconfirmed via analysis by LC-MS (FIG. 2B, 2C). The feasibility of themethod was further confirmed by radiolabeling a protein—a single domainantibody fragment derived from an anti-Class II MHC antibody (VHH4;unpublished)—using U-¹⁴C glucose. The desired product was obtained asassessed by SDS-PAGE and autoradiography, confirming the installation of¹⁴C-glucose onto the protein of interest (FIG. 3).

Example 2. Non-Invasive Imaging of Class II MHC Positive Cells in aTumor Model Using Site-Specifically ¹⁸F-Labeled and ⁶⁴Cu-Labeled SingleDomain Antibodies

Non-invasive imaging of an immune response against a tumor remains ahighly desirable and challenging goal. To this end, a labeling strategywas developed to generate a site-specifically ¹⁸F-labeled single domainantibody specific for murine Class II MHC products, ¹⁸F-VHH7. The¹⁸F-VHH7 product was used to perform positron emission tomography (PET)imaging in mice, using wild type, MHC II^(−/−) and NOD-SCID micexenografted with human melanoma as targets. Not only was ¹⁸F-VHH7rapidly cleared from the circulation, (t½<20 min), it also stainedsecondary lymphoid organs with remarkable specificity. Moreover, ClassII MHC positive cells surrounding a melanoma xenografted into nude micewere clearly imaged with ¹⁸F-VHH7, which enables the possibility ofearly detection of inflammatory cells as an indicator of disease.

Materials and Methods Synthesis of (Gly)₃-Tetrazine.

The tetrapeptide GGGC was synthesized by standard solid phase peptidesynthesis. Maleimide-tetrazine (from ClickChemistryTools) was dissolvedin 0.1 M phosphate buffer (PB) pH 7. The tetrapeptide GGGC was added andleft to stir at room temperature for 3 h until TLC (1:1 Hex:EtOAc v/v)indicated near-complete conversion to the product. The solution wasfiltered and purified by reverse phase-HPLC with a semi-preparativecolumn (Phenomenex, C₁₈ column, Gemini, 5 μm, 10×250 mm) at a flow rateof 5.0 mL/min; solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN.(G)₃-Tetrazine eluted at 30-35% solvent B. Fractions containing pureproduct were collected and lyophilized. LC-MS calculated forC₃₇H₅₄N₁₁O₁₃S[M+H]⁺ 892.362, found 892.370.

Synthesis of (Gly)₃-NOTA.

Maleimide-NOTA (from Macromolecules) was dissolved in 0.1 M PB pH 7. Thetetrapeptide GGGC was added at room temperature for 3 h until TLC (1:1Hex:EtOAc v/v) indicated almost complete conversion to the product. Thesolution was purified by RP-HPLC on a semi-preparative column(Phenomenex, C₁₈ column, Gemini, 5 μm, 10×250 mm) at a flow rate of 5.0mL/min; solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN. Thedesired product eluted from 15-20% solvent B. Fractions containing pureproduct were collected and lyophilized. LC-MS calculated forC₂₇H₄₅N₁₀O₁₁S [M+H]⁺717.298, found 717.305.

Enzymatic Incorporation of Substrates into Proteins Using Sortase.

The penta-mutant sortase A, with an improved k_(cat), was used (Chen,I., et al., “A general strategy for the evolution of bond-formingenzymes using yeast display.”, Proc. Natl. Acad. Sci. U.S.A. 2011; 108,11399-11404; the entire contents of which are hereby incorporated byreference). Reaction mixtures (1 mL) contained Tris.HCl (50 mM, pH 7.5),CaCl₂ (10 mM), NaCl (150 mM), triglycine-containing probe (500 μM),LPETG-containing probe (100 μM), and sortase (5 μM) (Theile, C. S., etal., “Site-specific N-terminal labeling of proteins usingsortase-mediated reactions.”, Nat. Protoc. 2013; 8, 1800-1807; Witte, M.D., et al. “Preparation of unnatural N-to-N and C-to-C proteinfusions.”, Proc. Natl. Acad. Sci. 2012; 109, 11993-11998; the entirecontents of which are hereby incorporated by reference). Afterincubation at 4° C. with agitation for 2 h, reaction products wereanalyzed by LC-MS, with yields generally >90%. When the yield was below90%, the reaction was allowed to proceed for an additional two hours,with addition of sortase to 10 μM and triglycine-containing probe to 750μM. Ni-NTA beads were added to the reaction mixture with agitation for 5min at 25° C. followed by centrifugation to remove sortase and anyremaining unreacted His-tagged substrate. The final product-either thetetrazine-labeled protein or NOTA-labeled protein, was purified by sizeexclusion chromatography in PBS or Tris.HCl (50 mM, pH 7.5).

Synthesis of ¹⁸F-TCO.

2-[¹⁸F]-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (¹⁸F-TCO) was prepared asdescribed (Keliher, E. J. et al. ChemMedChem 2011, 6, 424-427;incorporated herein by reference). [¹⁸F]-Fluoride (no carrier added,(n.c.a.)) in H₂ ¹⁸O, purchased from PETNET, was transferred to amicrowave reaction vessel (10 mL) and diluted with Kryptofix 2.2.2 (33mM in 300 μL MeCN) and K₂CO₃ (33 mM in 300μ L H₂O) solutions. The[¹⁸F]—F/K222/K₂CO₃ solution, 87.3±22.6 mCi (3230.1±836.2 MBq), was driedby azeotropic distillation of water with MeCN (added at 2, 6 and 8 min)by microwave heating (98° C., 150 W, 15 min) under a stream of argon.After drying, (E)-2-(cyclooct-4-enyloxy)ethyl 4-methylbenzenesulfonate(4 mg, 30 mmol) in DMSO was added, the vessel was sealed and thereaction heated by microwave (75 W) to 90° C. for 10 min. After coolingto 50° C., the mixture was diluted with MeCN (150 μL) and H₂O (750 μL)and subjected to preparative HPLC purification (1:1 MeCN/H₂O, 0.1%formic acid at 5.5 mL/min using a Macherey-Nagel Nucleodur C18 Pyramid10×250 mm Vario-Prep column). ¹⁸F-TCO was collected (t_(R)=13.5 min) in5-6 mL of solvent, diluted with H₂O (40 mL) and isolated by manual C18solid phase extraction. Elution from the C18 cartridge with DMSO (4×200μL) gave 22.1±4.0 mCi (817.6±149.5 MBq), a 35.6±4.9% decay-correctedradiochemical yield.

Synthesis and Characterization of ¹⁸F-VHHs.

In a typical reaction, a 1.5-mL centrifuge tube was loaded with VHH7-Tzin 1×PBS (40 μL, 150 μM), 1×PBS (300 μL), and ¹⁸F-TCO in DMSO (4.0 mCi(148.0 MBq), 100 μL). The tube was sealed and shaken at room temperaturefor 20 min. The mixture was analyzed by radio-TCO (ITLC, 100% MeCN,R_(f) ¹⁸F-TCO=0.9, R_(f) ¹⁸F-VHH7=0.0) showing 90% conversion to¹⁸F-VHH7. The reaction mixture was loaded onto a PD-10 size-exclusioncartridge (GE Healthcare) and elution with 1×PBS provided 2.3 mCi (85.1MBq) of ¹⁸F-VHH7 in 75.8% decay-corrected radiochemical yield. Startingwith 5.3 mCi (196.1 MBq) ¹⁸F-TCO, ¹⁸F-VHHDC13 was prepared following thesame procedure as described for ¹⁸F-VHH7 to give 2.8 mCi (103.6 MBq)after size-exclusion chromatography, a 69.7% decay-correctedradiochemical yield.

Synthesis and Characterization of ⁶⁴Cu-VHHs.

In a typical reaction, a 1.5-mL centrifuge tube was loaded withVHH7-NOTA (400 μL, 20 μM in 200 mM NH₄OAc buffer (pH 6.5)) and ⁶⁴CuCl₂(5.7 mCi, 210.8 MBq) in 200 mM NH₄OAc buffer (75 uL, pH 6.5). The tubewas sealed and shaken at 37° C. for 20 min. The mixture was analyzed byradio-TCO (ITLC, 50 mM EDTA pH 7, R_(f) ⁶⁴Cu/EDTA=1.0, R_(f)⁶⁴Cu-VHH7=0.0) showing 98% conversion to ⁶⁴Cu-VHH7. At this time themixture was loaded onto a PD-10 size-exclusion cartridge and elutionwith 1×PBS provided 5.2 mCi (192.4 MBq) of ⁶⁴Cu-VHH7 in 94.2%decay-corrected radiochemical yield. Starting with 3.5 mCi (129.5MBq)⁶⁴CuCl₂, ⁶⁴Cu-VHHDC13 was prepared following the same procedure asdescribed for ⁶⁴Cu-VHH7 to give 3.1 mCi (114.7 MBq) after size-exclusionchromatography, a 92.3% decay-corrected radiochemical yield. Prior toinjection both ⁶⁴Cu-VHH7 and ⁶⁴Cu-VHHDC13 were analyzed by radio-TLC(ITLC, 50 mM EDTA pH 7, R_(f) ⁶⁴Cu/EDTA=1.0, R_(f) ⁶⁴Cu-VHH7 and⁶⁴Cu-VHHDC13=0.0) and were found to have 99.6 and 99.8% radiochemicalpurity, respectively.

Pet-Ct Imaging.

All procedures and animal protocols were approved by the MassachusettsGeneral Hospital subcommittee on research animal care. For all imagingexperiments, mice were anesthetized using 1.5% isoflurane in 02 at aflow rate of ˜1 L/min. Mice were imaged with PET-computed tomography(CT) using an Inveon small animal scanner (Siemens, Munich, Germany).Each PET acquisition took approximately 30 minutes. A high-resolutionFourier rebinning algorithm was used to rebin sinograms, followed by afiltered back-projection algorithm to reconstruct three-dimensionalimages without attenuation correction. Isotropic image voxel size was0.796×0.861×0.861 mm, for a total of 128×128×159 voxels. Peaksensitivity of the Inveon accounts for 11.1% of positron emission, witha mean resolution of 1.65 mm. More than 100 counts were acquired perpixel, and the mean signal-to-noise ratio was greater than 20. CT imageswere reconstructed from 360 cone-beam x-ray projections with a power of80 keV and 500 μA. The isotropic resolution of the CT images was 60 μm.Reconstruction of data sets, PET-CT fusion, and image analysis were doneusing IRW software (Siemens). Two- and three-dimensional visualizationswere produced using the DICOM viewer OsiriX (The OsiriX Foundation,Geneva, Switzerland).

Blood Half-Life Measurement of ¹⁸F-VHHs.

Mice were administered 30±3 uCi of ¹⁸F-VHH7 by intravenous tail-veininjection. Blood samples were obtained by retro-orbital puncture usingtared, heparinized capillary tubes. Blood samples and capillaries wereweighed and radioactivity was measured using a Perkin-Elmer WallacWizard 3″ 1480 Automatic Gamma Counter. Non-linear regression analysiswas performed using GraphPad Prism 4.0c. Values are expressed aspercentages of the injected dose per gram of tissue and were fit to abi-exponential decay model. Data is shown in FIG. 6C.

Biodistribution Analysis of 18F- or 64Cu-VHHs.

Mice were administered 296±19 uCi of labeled VHHs by intravenoustail-vein injection. At 2 h post-injection, mice were euthanized,perfused with 1×PBS (20 mL), and dissected. Blood, urine and tissueswere excised and their wet weight was determined. Tissue radioactivitywas measured with a Perkin-Elmer Wallac Wizard 3″ 1480 Automatic GammaCounter. Statistical analysis was performed using GraphPad Prism 4.0c.Values are expressed as percentages of the injected dose (excretionsubtracted) per gram of tissue.

Results and Discussion

A challenge for immuno-PET using ¹⁸F as the tracer is its shorthalf-life (t_(1/2)=110 minutes), requiring it to be used almostimmediately after production. The approach exemplified here isreproducible and site-specific, without compromising the VHH's bindingsite, and is applicable to any other suitably modified biological entitysuch as cytokines. All of these steps—up to and including purificationof the desired product—are compatible with the short half-life of ¹⁸F(˜110 min.).

A facile two-step process was developed for labeling proteins equippedwith a sortase recognition motif. The ¹⁸F-radionuclide was conjugated tothe VHHs via the tetrazine (Tz)/trans-cyclooctene (TCO) reverse-electrondemand Diels-Alder cycloaddition reaction. The TCO-Tetrazine reaction isthe fastest known bioorthogonal reaction to date, with an estimatedsecond order rate constant of 2000±400 M⁻¹ s⁻¹ (Blackman, M. L. et al.Am. Chem. Soc. 2008, 130, 13518-13519; which is incorporated byreference). The requisite sortase nucleophile, GGG-tetrazine, whichparticipates efficiently in sortase reactions, was synthesized in nearlyquantitative yields. Thus, using ¹⁸F-TCO, prepared via a previouslypublished method, (Keliher, E. J. et al. ChemMedChem 2011, 6, 424-427;incorporated herein by reference) >5 mCi of ¹⁸F-labeled VHH7 was readilyproduced in two steps.

A tosyl-trans-cyclooctene (TCO) was reacted with ¹⁸F—NaF for ˜10 min toproduce ¹⁸F-TCO. The product was purified via HPLC. Next, ¹⁸F-TCO wasadded to the Tz-modified protein, and the reaction was allowed toproceed for ˜20 min at pH 7. The ¹⁸F-labeled protein product was thenquickly purified via a PD-10 column pre-equilibrated with PBS, providinga radiolabeled protein solution ready for injection. The labelingexperiments with ¹⁸F-TCO yielded ¹⁸F-VHH7 in 61±9% decay-correctedradiochemical yield (FIG. 4).

Antibody labeling strategies for PET often revolve around modificationwith metal chelators to enable installation of radioisotopes such as⁶⁴Cu, ⁶⁸Ga, or ⁸⁹Zr (Tanaka, K. et al. “PET (positron emissiontomography) imaging of biomolecules using metal-DOTA complexes: a newcollaborative challenge by chemists, biologists, and physicians forfuture diagnostics and exploration of in vivo dynamics.”, Org. Biomol.Chem. 2008; 6, 815-828; Fani, M., et al. “68Ga-PET: a powerfulgenerator-based alternative to cyclotron-based PETradiopharmaceuticals.”, Contrast Media Mol. Imaging 2008; 3, 53-63;Vosjan, M. J. W. D. et al. “Conjugation and radiolabeling of monoclonalantibodies with zirconium-89 for PET imaging using the bifunctionalchelate p-isothiocyanatobenzyl-desferrioxamine.”, Nat. Protoc. 2010; 5,739-743; the entire contents of each are hereby incorporated byreference). The longer half-life of ⁶⁴Cu (12.7 h) relative to ¹⁸F (110min) in principle allows a more extended imaging period to establishtissue penetration and dwell time of VHHs on their targets. ANOTA-(Gly)₃ sortase nucleophile was developed to enable site-specificlabeling of VHHs via this high affinity copper chelating agent (K_(eq)for Cu²⁺: ˜10²¹) (Delgado, R. et al. “Stabilities of divalent andtrivalent metal ion complexes of macrocyclic triazatriacetic acids.”,Inorg. Chem. 1999; 32, 3320-3326; the entire contents of which arehereby incorporated by reference). The [Cu²⁺-NOTA] complex iskinetically inert, with minimum metal exchange when exposed to othermetals present in body (Zhang, Y. et al. “Positron Emission TomographyImaging of CD105 Expression with a 64Cu-Labeled Monoclonal Antibody:NOTA Is Superior to DOTA.”, PLoS ONE. 2011; 6, e28005; the entirecontents of which are hereby incorporated by reference).Site-specifically labeled [(⁶⁴Cu)-NOTA]-VHHs were produced in highradiochemical yield (˜90% decay corrected) (FIGS. 4E, 4F and 4H) andused ⁶⁴Cu-VHH7 to image a C57BL/6 mouse at 4 h, 8 h, and 24 h postinjection. VHH7 stayed on its target (secondary lymphoid organs) evenafter 24 hours (FIG. 8) but produced images with an inferior signal tonoise ratio when compared with ¹⁸F-VHH7.

Fluorophores were installed in a sortase-catalyzed reaction, and thesefluorescently labeled VHHs were used as staining agents forcytofluorimetry, establishing that VHH7 recognizes murine I-A productsencoded by the H-2^(b) and H-2^(d) haplotypes present in the commonlaboratory strains of mice. Administration of ¹⁸F-VHH7 in vivo showedexcellent visualization of normal lymph nodes, spleen and thymus with ahigh degree of specificity and high signal to noise ratios observed inMHC-II⁺ mice (C57BL/6). Very little if any specific labeling is observedin MHC-II deficient mice (B6.12952-H2<dlAb1-Ea>/J); (FIG. 5).

¹⁸F-VHH7 was also used for biodistribution analysis and blood half-lifemeasurements in a C57BL/6 mouse model (FIG. 6). Results show a t_(1/2)of <20 min and excellent specificity of VHH7 for normal lymph nodes,spleen and thymus in MHC-II⁺ mice (C57BL/6). Very little specificlabeling is observed in MHC-II deficient mice.

The application of anti-class II MHC single domain antibodies was alsoexplored to determine whether it is feasible to image the behavior oflymphocytes in a xenograft tumor model. Both the distribution ofmacrophages at steady state and the evolution of their distribution andlocation over time in the course of such a response have never beforebeen imaged non-invasively. The ability to monitor the presence orabsence of macrophages endowed with markers such as Class II MHC duringthe course of clinical treatment is particularly important. Theseapproaches are transposable to a clinical setting, because VHHs of thistype, after suitable modification (humanization), have been used inPhase I and Phase II clinical trials for therapeutic indications (DeMeyer, T. et al. Trends Biotechnol. 2014, 32, 263-270; which isincorporated by reference). The ability to assess the presence anddistribution of the immunological checkpoint molecules presents anenormous enrichment of the diagnostic toolbox with which to monitor—ifnot predict—the clinical behavior of the appropriate patientpopulations.

In many tumor models, the margins of the tumor contain macrophages,which if activated should express Class II MHC products. Thus, PETimaging experiments were designed to investigate whether, in addition tothe usual lymphoid structures as visualized by radiolabeled VHH7, it wasalso possible to detect the presence of macrophages around and possiblywithin a xenografted tumor. The Mel-Juso human melanoma cell line ispositive for human Class II MHC products, and has been extensivelycharacterized with respect to trafficking and surface display of ClassII MHC molecules (Tulp, A. et al. Nature 1994, 369, 120-126; Bakke, O.;et al. Cell 1990, 63, 707-716; which are incorporated by reference). Assuch, it is representative of a subset of human melanomas. Accordingly,Mel-Juso xenografts were created in NOD-SCID mice by subcutaneousinoculation. The SCID mice hosts contain some of the normal complementof murine Class II MHC⁺ antigen presenting cells such as macrophages,but no B or T cells, and should thus still be visible upon PET imagingwith radiolabeled VHH7. Result showed that by using ¹⁸F-VHH7,inflammation around the tumor is clearly visible (FIG. 7A-D). Thisimportant observation shows the possibility of early state detection ofdiseases such as multiple sclerosis (MS), diabetes, and cancers usingthis methodology.

To determine if it is possible to image small tumors at earlier stagesof growth NOD/SCID mice xenografted with 5×10⁶ Mel-Juso human melanomacells (FIG. 7 E-J) were imaged at 6 days (FIG. 7F), 20 days (FIG. 7E)and 27 days post-injection Inflammation was detected at the site of themalignant growth as the earliest time point after injection, a time whenthe incipient tumors are not detectable by palpation, only bycytofluorimetry or histology (FIG. 71). Cytofluorimetry on cellsuspensions prepared from the excised tumors again confirmed thepresence of tumor-infiltrating Class II MHC⁺ cells. It may thus beworthwhile to explore early stage detection of diseases such as MS,diabetes, different infections or cancers, all characterized by aninflammatory signature (Keliher, E. J., et al. “High-Yielding, Two-Step¹⁸F Labeling Strategy for ¹⁸F-PARP1 Inhibitors.”, ChemMedChem, 2011; 6,424-427; which are incorporated by reference).

Inflammation in response to administration of complete Freund's adjuvant(CFA) was also examined using ¹⁸F-labeled VHH7 and VHH DC13.Subcutaneous administration of CFA into one of the front paws resultedin inflammation, imaged 24 h post-injection using ¹⁸F-labeled VHH7 andVHH DC13, a single domain antibody that recognizes the neutrophil,macrophage and dendritic cell marker CD11b, as determined by massspectrometry of immunoprecipitates prepared with immobilized VHH DC13.VHHs revealed inflammation with remarkable selectivity, with VHH DC13showing a stronger signal in the inflamed region relative to VHH7 (FIG.9 A&B). Selective accumulation of ¹⁸F VHHDC13 when compared with thebuild-up of ¹⁸F VHH7 is consistent with the massive influx of CD11b⁺neutrophils generally observed at the site of injection 24 h afteradministration of CFA. It is thus possible to monitor inflammation andimmune responses non-invasively by exploiting specific sentinels such asClass II MHC⁺ or CD11b⁺ cells. A VHH was generated that recognizes humanHLA-DR molecules in a monomorphic pattern of reactivity. The ability tomonitor the presence or absence of activated macrophages as an indicatorof inflammation will therefore be transposable to a clinical setting asa diagnostic tool.

In conclusion, exemplified here is the first enzyme mediatedsite-specific ¹⁸F-labeling and ⁶⁴Cu-labeling of proteins using sortase.The method is highly efficient and compatible with the short half-lifeof ¹⁸F. Using this method, ¹⁸F-VHH7, the camelid-derived single domainantibody against murine MHC class II molecules, was produced andsuccessfully used to visualize and map the secondary lymphatic organsincluding lymph nodes, thymus and spleen. Results showed that not onlyis VHH7 rapidly cleared from the circulation (t ½<20 min), but alsostained the secondary lymphoid organs with excellent specificity. Thismethod thus represents a significant new tool for non-invasive imagingof the lymphatic system. Next, the application of the method to imageinflammation was explored. Accordingly, xenograft mice models bearinghuman melanoma tumors were created and imaged with ¹⁸F-VHH7.Importantly, due to the infiltration of macrophages, it was possible toclearly see the inflammation around the tumor. Thus, this method isamenable for early detection of many diseases such as MS, diabetes anddifferent types of cancers. Further, the methodology exemplified herecan be used with other proteins and VHHs for PET imaging. For example,the anti-mouse CD3 VHH can be used to visualize CD4⁺ and CD8⁺ T cells aswell as CD4⁺CD8⁺ thymocytes. These experiments can be performed inRAG2−/− mice as the negative control (complete absence of T cells) andin C57B/6 mice as the positive control. A comparison of the VHH7 and CD3VHH should prove informative on the ability to distinguish between theClass II MHC-positive localization (antigen presenting cells, B cells)and T cell localization. Additionally, the anti-CD11b VHH can be used todistinguish between B cells and macrophages.

Example 3. ¹⁸F-Labeling of Proteins Using 18F-FDG Generated from aCatalyzed Oxime Ligation

An ¹⁸F-FDG was added to a solution of Tetrazine-aminooxy and a catalystto generate the ¹⁸F labeled click chemistry handle (¹⁸F-FDGoxime-tetrazine) by oxime ligation (FIG. 10A). A series of catalysts,including m-phenylenediamine, p-phenylenediamine and p-anisidine weretested to optimize this reaction. The data showed thatp-phenylenediamine (pPDA) was the most efficient catalyst for thereaction. The optimized condition for this reaction is as follows:[Tz-Aoxy]=350 mM, [pPDA]=400 mM, T=75° C. HPLC showed >95% conversion tothe product in approximately 5 minutes.

Following the oxime ligation reaction, the product is purified via HPLCto provide a 18F-labeled Tetrazine. The fraction from HPLC is mixed with4× volume of 1% NaHCO3 and is loaded into a solid phase extraction (SPE)column. The 18F-tetrazine will stick to the column and hence isconcentrated. The 18F-tetrazine is eluted with pure acetonitrile orethanol (or a similar organic solvent) from the SPE column to a finalvolume of ˜1 mL. This can be further dried or be used as it is dependingto the scale of the nanobody conjugation in the next step. The organicsolvent can be up to ˜25% of the volume for the final conjugationreaction and not higher. The ¹⁸F-FDG oxime-tetrazine product can then beused to label a pre-prepared sortagged VHH-TCO to form the final¹⁸F-labeled VHH as shown in FIG. 10C.

All publications, patents and sequence database entries mentionedherein, including those items listed above, are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the claims or from relevant portions of the description isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

1-27. (canceled)
 28. A composition comprising a radiolabeled sortasesubstrate polypeptide, a sortase, and a protein comprising a sortaserecognition motif.
 29. The composition of claim 28, wherein the sortasesubstrate polypeptide comprises an N-terminal sortase recognition motif.30. The composition of claim 29, wherein the N-terminal sortaserecognition motif comprises an oligoglycine or an oligoalanine sequence.31. The composition of claim 30, wherein the oligoglycine or theoligoalanine comprises 1-10 N-terminal glycine residues or 1-10N-terminal alanine residues, respectively.
 32. The composition of claim31, wherein the N-terminal sortase recognition motif comprises thesequence GGG.
 33. (canceled)
 34. The composition of claim 28, whereinthe sortase substrate polypeptide is linked to a radiolabeled agentlabeled with a radionuclide that is carbon-11, carbon-14, nitrogen-13,oxygen-15, fluorine-18, rubidium-82, copper-61, copper-62, copper-64,yttrium-86, gallium-68, zirconium-89, or iodine-124.
 35. The compositionof claim 34, wherein the radiolabeled agent is labeled with aradionuclide that is fluorine-18.
 36. The composition of claim 35,wherein the radiolabeled agent comprises a sugar.
 37. The composition ofclaim 36, wherein the sugar is fludeoxyglucose (¹⁸F-FDG).
 38. (canceled)39. The composition of claim 28, wherein the sortase is sortase A fromStaphylococcus aureus (SrtA_(aureus)), sortase A from Streptococcuspyogenes (SrtA_(pyogenes)), sortase B from S. aureus (SrtB_(aureus)),sortase B from Bacillus anthracis (SrtB_(anthracis)), or sortase B fromListeria monocytogenes (SrtB_(monocytogenes)).
 40. The composition ofclaim 28, wherein the protein is an antibody, an affibody, asingle-domain antibody, a Fab fragment, or a therapeutic peptide. 41.The composition of claim 40, wherein the protein binds to a tumor cell,a tumor-associated cell, a tumor antigen, or to a marker ofinflammation.
 42. (canceled)
 43. The composition of claim 28, whereinthe protein comprises a C-terminal sortase recognition motif.
 44. Thecomposition of claim 43, wherein the C-terminal recognition motif isLPXTX; LPETG (SEQ ID NO:2); LPETA (SEQ ID NO:3); NPXTX; NPQTN (SEQ IDNO:4); or NPKTG (SEQ ID NO:5); wherein each instance of X independentlyrepresents any amino acid residue. 45-47. (canceled)
 48. A compositioncomprising a radiolabeled protein generated using the method of claim 1.49-57. (canceled)
 58. A method for radiolabeling a sortase substratepeptide comprising contacting a sortase substrate peptide that comprisesa nucleophilic group with a radiolabeled agent that comprises anelectrophilic group under conditions suitable for the formation of acovalent bond between the sortase substrate peptide and radiolabeledagent. 59-116. (canceled)
 117. A method of diagnosing, monitoring, ortreating a subject comprising: (a) administering the composition ofclaim 48 to the subject; and (b) detecting the radiolabel in thesubject. 118-120. (canceled)
 121. A method for site-specificallyradiolabeling a protein comprising: contacting a radiolabeled agentconjugated to a first click chemistry handle with a protein having asecond click chemistry handle to produce a site-specificallyradiolabeled protein. 122-222. (canceled)
 223. A radioactive protein ofFormula (II)

wherein L¹ is a linker comprising at least four amino acids formed byenzymatic conjugation between two enzyme recognition sequences; and L²is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene. 224-320.(canceled)
 321. A pharmaceutical composition comprising the radioactiveprotein of claim 223 and a pharmaceutically acceptable carrier. 322.(canceled)